Streptococcus suis, a Gram-positive bacterium, is an important swine and human pathogen, with serotype 2 being the most prevalent strain found worldwide. Deafness, meningitis, and death (in severe cases) are observed in S. suis-infected cases. Development of the ligands that can bind to S. suis with high affinity and specificity could be beneficial for the diagnosis and treatment of S. suis infection. Herein, the nuclease-resistant RNA aptamers based on 2′-fluoropyrimidine modification against S. suis serotype 2, strain P1/7, were established using the cell- Systematic Evolution of Ligands by Exponential enrichment (SELEX) technique. One of the aptamers, R8-su12, could bind to the S. suis target strain as well as other S. suis serotypes, i.e., 1, 1/2, 9, and 14, but not to other bacteria tested, i.e., S. pneumoniae ATCC 49619, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853. Moreover, the R8-su12 RNA aptamer was also capable of inhibiting the biofilm formation of the S. suis target strain, making it potentially useful for the study of biofilm formation and the treatment of S. suis infection in humans and pigs in the future. Streptococcus suis is a zoonotic pathogen causing infectious diseases in pigs and humans such as meningitis, pericarditis, and septic shock [S. suis has been classified into 29 serotypes (SS) based on the specific antigens that appear on the polysaccharide capsule [S. suis SS 2 infection was the most prevalent human case in Thailand, especially in the northern region [S. suis are culture and biochemical tests [S. pneumoniae, S. bovis, enterococci, viridans streptococci or even Listeria monocytogenes [S. suis is biofilm formation [S. suis pathogenesis is needed for the effective control of S. suis infection in humans and animals.ic shock ,2. To da capsule ,4. S. sun region ,6. Conveal tests , which aytogenes . This caormation , which aormation . An undeS. suis with high affinity and specificity for research, diagnostic, and therapeutic purposes. One example is aptamers, which are single-stranded DNA or RNA [S. suis-specific RNA aptamers were developed because RNA has a greater ability to fold into more diverse three-dimensional conformations, which are capable of binding to target molecules, compared with DNA [2), or methoxy (-OMe) groups [Different types of ligands could be generated to bind A or RNA . The aptA or RNA ,14. In twith DNA ,16. In a) groups . S. suis SS 2, type strain P1/7 were successfully generated using the whole-cell SELEX approach. One of them, the R8-su12 RNA aptamer, could specifically recognize S. suis and inhibit biofilm formation, making it potentially useful for the study of biofilm formation and the treatment of S. suis infections in humans and pigs in the future.In this study, nuclease-resistant RNA aptamers against S. suis SS 2, P1/7, approximately 1014 random RNA library pool was produced. A total of 10 mg/mL baker’s yeast tRNA was also added to the library pool as a binding competitor to reduce non-specific RNA binding. Then, the random RNA library mixture was incubated with 107 live S. suis SS 2, P1/7 cells. The cell-SELEX was performed with a total of eight rounds. The enrichment of aptamer pool during selection rounds 4 (R4), 6 (R6), and 8 (R8) were measured using real-time reverse transcription (RT) qPCR compared with the random RNA library (Lib). The fold change in the relative binding affinity of RNA aptamer was increased in the later round of cell-SELEX (data not shown). The latest R8 RNA aptamer pool was used to determine the relative binding affinity compared to the starting N40 Lib. After calculation, the % binding of R8 RNA aptamer pool was higher than % binding of Lib with statistical significance (p = 0.023), implying the enrichment of the R8 RNA aptamer pool against S. suis SS 2, P1/7   B. These Therefore, the aptamer pool from R8 was cloned and sequenced. According to the sequencing analysis of 26 clones, they can be classified into four distinct groups as shown in S. suis SS2, P 1/7. The representative R8-su12 RNA aptamer from group 1 was then selected for the prediction of secondary structures; the folding temperature was set at 37 °C. The R8-su12 RNA aptamer had one predicted secondary structure with a folding energy  equal to −24.30 kcal/mol. The predicted secondary structure consists of an external loop with four bases and one closing helices. Moreover, loops and stems were also found, as shown in Thus, group 1 showed the highest consensus sequences frequency among the 26 clones, suggesting that they might have a high affinity toward S. suis SS 2, P1/7 and non-target cells including the American Type Culture Collection (ATCC) strains of S. pneumoniae, S. aureus, E. coli, and P. aeruginosa, were evaluated. The negative control—L2 RNA aptamer (5′-GGGAGUCGACCGACCAGAAGAAGCUGCUUCAAAAUAGAUCUACACUCACAUAAGGCGAAUAUGUGCGUCUACAUCUAGACUCAU-3′) was used in this experiment, instead of a random RNA Lib. The R8-su12 and L2 RNA aptamer were incubated with target and non-target cells. As shown in S. suis SS 2, P1/7 when compared with S. pneumoniae, S. aureus, E. coli, and P. aeruginosa. . To test the specificity of the R8-su12 RNA aptamer, the target S. suis SS 1/2, 1, 9, and 14. These strains are closely related to the target cells and showed a high prevalence of S. suis infection strains in Thailand [S. suis strains. Interestingly, the R8-su12 RNA aptamer showed the highest % binding when tested with S. suis SS 1/2, followed by S. suis SS 1, 2, 14, and 9, respectively (S. suis SS 1 (p = 0.021) compared to the target cells. On the other hand, a lower binding affinity against S. suis SS 9 (p = 0.021) was shown. No significant binding was found in S. suis SS 1/2 (p = 0.275) and S. suis SS 14 (p = 0.127)  B.S. suis [S. suis cells might affect the biofilm formation. As shown in the result, the biofilm formation of S. suis SS 2, P1/7 cultured with R8-su12 RNA aptamer, L2 RNA aptamer and baker’s yeast tRNA was significantly reduced by 61.2%, 12.8% and 12.9 %, respectively, when compared to control (p < 0.05). Noticeably, the influential activity that decreased biofilm formation was also observed in R8-su12 RNA aptamer treated S. suis SS 2, P1/7, showing 55.5% and 55.4% reductions when compared to L2 RNA aptamer (p < 0.001) and baker’s yeast tRNA (p < 0.001), respectively. While the presence of L2 RNA aptamer and baker’s yeast tRNA in the culture medium, biofilm formation showed no statistically significant difference (p = 0.988)  . These rS. suis is reported as a zoonotic pathogen causing infectious diseases in pigs and humans, such as meningitis, pericarditis, and septic shock [S. suis serotype 2 infection is the most prevalent human case in the world, including Thailand, especially in the northern region [S. suis infection is needed. The development of ligands that can bind to S. suis with high affinity and specificity could be beneficial for the diagnosis and treatment of S. suis infection. ic shock ,2. S. sun region ,6. NowadS. suis SS 2, P1/7 was first established. RNA aptamers were chosen over DNA aptamers for their ability to fold into a more diverse, three-dimensional structures [S. suis cell surface show high levels of similarity [S. suis remained in the sample well with bacterial cells. Theoretically, the active separation using electrophoresis would remove non-binding RNAs from the target cells more efficiently than the washing separation, which relies on the passive dissociation of RNAs. Therefore, the stringency of selection was increased, resulting in the successful recovery of the high-affinity S. suis aptamers.In this study, the nuclease-resistant RNA aptamer, R8-su12, against ructures ,16. Moreructures ,21,22. Tmilarity . This apmilarity . In addiS. suis SS 2, P1/7, as well as the others S. suis serotypes, 1, 1/2, 2, and 14. Unfortunately, the target molecule of the aptamer was not determined in this study. We speculated that the potential targets of the R8-su12 RNA aptamer could be some components of capsular polysaccharides (CPSs) that are shared between these serotypes. There are several studies revealed that S. suis serotypes 1, 1/2, 2, and 14 contain sialic acid in their CPSs [For the specificity determination, the data showed that the R8-su12 RNA aptamer bound to target cells, eir CPSs ,25,26. TS. suis is biofilm formation. This leads to an increased resistance to host defenses and antimicrobial agents, making pathogens difficult or impossible to eliminate [S. suis biofilm formation. We found that the derived aptamer can reduce the biofilm formation of S. suis SS 2, P1/7. The studies on the bacteria biofilm revealed that the regulatory mechanism can be divided into eight categories, depending on the different communication signaling molecules produced by bacteria [S. suis biofilm formation. The study also showed that the ability of the complementary mutant (CΔcysM) strain to form a biofilm may indirectly related to the QS system. It is possible that the R8-su12 RNA aptamer might bind to those molecules, indirectly or directly playing a role in biofilm formation. However, additional studies are required to investigate how the aptamer affects biofilm formation.One of the important virulence characteristics of liminate . Therefobacteria . These cbacteria  revealedStreptococcus suis serotype 2, type strain P1/7   was used as the target cell in this study. For specificity testing, other bacterial species, including Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Streptococcus pneumoniae ATCC 49619, S. pyogenes ATCC 19615, S. suis serotype 1 DMST, S. suis serotype 1/2 DMST, S. suis serotype 14 DMST, and S. suis serotype 9 clinical isolates were used. S. suis SS 2, P1/7 was cultured onto 5% blood agar (BA)  and incubated at 37 °C, 5% CO2 for 18–24 h. For other bacterial species were incubated overnight for 18–24 h at 37 °C, 5% CO2 on 5% BA  or trypticase soy agar  depending on their optimal medium. To prepare the target cells for the SELEX process, fresh colonies from BA  were harvested and washed thrice with 1 mL of Phosphate Buffer Saline (PBS). Then, the bacterial cells were adjusted to reach at the desired OD600 with a total of 1 mL of binding buffer before being loaded into a centrifugal filter .The The starting 2′-F pyrimidine modified RNA Lib was prepared by in vitro transcription of a N40 DNA Lib containing a 40-nt random region and primer binding sites (5′-AGTAATACGACTCACTATAGGGAGTCGACCGACCAGAA-N40-TATGTGCGTCTACATCTAGACTCAT-3′) using T7 R&D polymerase (50 U/µL) .14 molecules, 100 pmol of random RNA Lib was used in the first round of cell-SELEX. The 2′-F random RNA Lib was thermally equilibrated for proper folding in a total volume of 100 µL binding buffer  with 10 mg/mL baker’s yeast tRNA  at 65 °C, followed by a cool-down at room temperature for 5 min. The freshly prepared 107 cells of S. suis SS 2, P1/7 were incubated with the equilibrated random RNA Lib at room temperature in the centrifugal filter for 60 min in rounds 1–7 and 50 min in round 8, with gentle rotation. The unbound or weakly bound aptamers were partitioned by washing in binding buffer for various specific volumes, numbers of washes, and incubation times on the rotator according to the selection protocol  and 3′-N40 primer (5′-ATGAGTCTA GATGTAGACGCACATA-3′). Then, the cDNA was amplified with Taq DNA polymerase  using 3′-N40 primer and 5′-primer containing T7 promoter (underlined) (5′-AGTAATACGACTCACTATAGGGAGTCGACCGACCAGAA-3′). The PCR products were transcribed in vitro, purified using the Qiaquick PCR Purification Kit , and used for the next round of cell-SELEX. The cell-SELEX was performed until the 8th round (R8).The bound RNAs were eluted using 100 µL of elution buffer , followed by ethanol precipitation. The RNA pool was reverse-transcribed using Superscript7 cells of S. suis SS 2, P1/7. Both Lib and Rx were saved for approximately 10 µL as input RNA. After binding, washing, and elution steps, as described above, the amount of input and bound RNAs were quantified using real-time RT qPCR. The real-time master mix was generated in a total 10 µL per reaction comprising the final concentration of 1× SensiFAST™ SYBR® No-ROX Kit , 0.5 µM of each 3′-N40 primer and 5′-primer (5′-GGGAGTCGACCGACCAGAA-3′), and 0.5 µL of cDNA from each RT reaction . Each sample was performed in triplicate. For non-template control (NTC), real-time PCR master mix and cDNA from NTC-RT reaction were used. The real-time RT qPCR was performed under the following conditions: 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, with a total of 45 cycles. The fluorescence signal was read at 60 °C and the association curve was set at 65 °C to 95 °C. The binding percentage was calculated using Formula (1):elute refers to the cycle threshold of elute sample and Ctinput refers to the cycle threshold of input sample. The % binding of random RNA library (% bindingLib) and the % binding of Rx (% bindingRx) were then calculated into the fold change using Formula (2):The enrichment of the aptamer pool in selected rounds (Rx), which were R4, R6, and R8, was quantified by relative binding affinity using the binding assay. One hundred picomoles of either the control RNA Lib or selected aptamer pool in the presence of baker’s yeast tRNA was incubated with approximately 10S. suis SS 2, P1/7 and non-target cells, including other species of Streptococcus , Gram-positive bacteria (S. aureus), Gram-negative bacteria , and other S. suis serotype . One hundred picomole of the control (random RNA Lib pool or L2 aptamer) and the derived nuclease-resistant RNA aptamers were incubated with 107 of target cells, S. suis SS 2, P1/7 and non-target cells. Then, the cell-SELEX was processed until the eluate was obtained. After that, it was used as a template in the binding assay using real-time RT qPCR. The binding percentage and fold change were calculated as described above.For specificity testing, the R8 aptamer pool and representative R8-su12 RNA aptamers were screened toward https://www.ebi.ac.uk/Tools/msa/clustalo/ accessed on 11 November 2020). The selected RNA aptamer was also predicted for secondary structure using the mfold Web Server (http://www.unafold.org/mfold/applications/rna-folding-form.php accessed on 7 February 2021) to determine the binding sites between the aptamer and target cells.The aptamer pool from cell-SELEX round 8 was cloned using pTG19-T PCR cloning vector B . Twenty-six clones were sequenced and analyzed for similarity using the Clustal Omega Multiple Sequence Alignment  of R8-su12 RNA aptamer with 10 mg/mL baker’s yeast tRNA, negative control L2 RNA aptamer with 10 mg/mL baker’s yeast tRNA, and 10 mg/mL baker’s yeast tRNA alone. Then, the mixture was transferred into a 24-well polystyrene plate containing 1.8 mL of BHI broth. The plates were incubated at 37 °C for 3 days without shaking. After staining with 0.1% crystal violet for 30 min, the OD600 was measured. The BHI broth with the supplements was performed as a negative control, and its OD600 was set as blank. These blank absorbance values and cut off values (ODc) were used to calculate and interpret the biofilm formation result as described [To examine the RNA aptamer’s ability to inhibit biofilm formation, the detection of biofilm formation was carried out according to Meng et al.  with somescribed . Each sap < 0.05 or 0.001. All the calculations were performed using the SPSS Statistics 6.0 software .All samples were tested in triplicate with at least three independent experiments. The one-way analysis of variance and Mann–Whitney U test were used to determine the statistically significant when S. suis SS 2, strain P1/7, R8-su12, was successfully constructed. Moreover, the derived RNA aptamer revealed the ability to bind to other pathogenic S. suis . Remarkably, the R8-su12 RNA aptamer can significantly reduce the biofilm formation, one of the S. suis virulence factors. However, the target ligand of the aptamer on the S. suis SS 2, strain P1/7 cell surface would be further identified. This might be useful for understanding the pathogenesis, leading to an early diagnostic, treatment, and effective control of S. suis infection.Herein, the nuclease resistant RNA aptamer specific to


DDX41, which encodes a DEAD-box type RNA helicase, are present in approximately 2–5% of AML and MDS patients; this disease subtype exhibits a distinctive disease phenotype characterized by late age of onset, tendency toward cytopenia in the peripheral blood and bone marrow, a relatively favorable prognosis, and a high frequency of normal karyotypes. Typically, individuals with a loss-of-function germline DDX41 variant in one allele later acquire the p.R525H mutation in the other allele before overt disease manifestation, suggesting that the progressive decrease in DDX41 expression and/or function is involved in myeloid leukemogenesis.RNA helicases play roles in many processes involving RNA metabolism by altering RNA structure and RNA-protein interactions through ATP-dependent helicase activity. A single RNA helicase can play multiple cellular roles, making it difficult to elucidate the mechanisms by which mutations in DDX41 are involved in leukemogenesis. Nevertheless, multiple DDX41 functions have been associated with disease development. The enzyme has been implicated in the regulation of RNA splicing, nucleic acid sensing in the cytoplasm, R-loop resolution, and snoRNA processing.Most of the mutated RNA splicing-related factors in MDS are involved in the recognition and determination of 3’ splice sites (SS), although their individual roles are distinct. On the other hand, DDX41 is likely incorporated into the C complex of the spliceosome, which may define a distinctive disease phenotype. This review summarizes the current understanding of how DDX41 is involved in this unique myeloid malignancy.In myeloid malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), patient selection and therapeutic strategies are increasingly based on tumor-specific genetic mutations. Among these, mutations in  In brief, Polprasert et al. performed a comprehensive genetic analysis of families with suspected inherited myeloid malignancies without known mutations such as RUNX1, CEBPA, and GATA2 and isolated DDX41 as a new disease-associated gene , respectively. They play multiple celluar roles including those involved with transcription, RNA splicing, ribosome biogenesis, and translation  and proteins in this class are categorized into the SF1 to SF6 superfamilies . The SF2nslation , 43, 44.nslation . Single nslation . Althougo groups . Specifio groups . In the Comprehensive analysis for factors that constitute the spliceosome at each phase of RNA splicing have suggested that DDX41 is a component of splicing C complex , 49. RNASF3B1, SRSF2 and U2AF1) all encode factors involved in the recognition of the 3’SS (DDX41 variants (with or without somatic DDX41 mutations), and 2 of 19 cases with somatic DDX41 mutations alone had mutations in at least one of the genes encoding typical MDS-related RNA splicing factors  with a variant allele frequency of 3% or more  has been reported to alter 3’SS selectivity  at its N-terminus, and recent studies revealed that it is predominantly detected in the nucleus . HoweverAn increase in R-loop formation occurs in MDS regardless of the mutation spectrum present in tumor cells , 70. R-lvia the STING-TBK1 axis  processing . The stuvia DDX41.However, this discovery simultaneously raises another question regarding the role that DDX41 plays in snoRNA processing. Although reduced expression of DDX41 impairs intron removal in certain host genes, it is still unclear how it confers such splicing selectivity. Considering that RNA splicing factors play a role in snoRNA processing, as shown for IBP160 , it is pDDX41 mutations are more likely to exhibit a DNA damage phenotype that renders them more prone to apoptosis than to proliferation, at least prior to overt disease development. DDX41 may play a role in late process of RNA splicing, and mutant-related defects would affect the transcription and DNA replication that normally progress in concert with RNA splicing. This hypothesis may explain the unique phenotypes of this disease. Minor clones with biallelic DDX41 mutations emerged from those with a germline variant and define the disease phenotype , No. 21K08419, and No. 18K08334 (HM) and No. 22K10195 (SS). This work was also partly supported by the Foundation for Promotion of Cancer Research.The authors would like to express our sincere appreciation to all those who supported our work, especially Ms. Kazue Akita and Ms. Aya Higashi in our laboratory.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


RNAs are rapidly degraded in samples and during collection, processing and testing. In this study, we used the same method to explore the half-lives of different RNAs and the influencing factors, and compared the degradation kinetics and characteristics of different RNAs in whole blood and experimental samples. Fresh anticoagulant blood samples were incubated at room temperature for different durations, RNAs were extracted, and genes, including internal references, were amplified by real-time quantitative PCR. A linear half-life model was established according to cycle threshold (Ct) values. The effects of experimental operations on RNA degradation before and after RNA extraction were explored. Quantitative analysis of mRNA degradation in samples and during experimental processes were explored using an orthogonal experimental design. The storage duration of blood samples at room temperature had the greatest influence on RNA degradation. The half-lives of messenger RNAs (mRNAs) was 16.4 h. The half-lives of circular RNAs (circRNAs), long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) were 24.56 ± 5.2 h, 17.46 ± 3.0 h and 16.42 ± 4.2 h, respectively. RNA degradation occurred mainly in blood samples. The half-life of mRNAs was the shortest among the four kinds of RNAs. Quantitative experiments related to mRNAs should be completed within 2 h. The half-lives of circRNAs and lncRNAs were longer than those of the former two. Compared with tissue samples, blood samples have advantages including being more readily accessible, suitable for continuous sampling, and amenable to numerous different tests. However, after blood samples are collected, they need to be processed, packed and stored in containers suitable for long-term cryopreservation. Otherwise, RNA in blood is prone to degradation under normal temperature transportation and daily storage conditions, which can affect the subsequent detection, analysis and development of molecular diagnostic biomarkers2.Blood samples are rich in biomolecules such as proteins, DNA and RNA, which can be used for disease diagnosis, and determining disease stage and prognosis3. As a basis for biomarker research and development, the acquisition of high quality blood samples is critical. The dipotassium ethylene diamine tetraacetic acid (EDTA-2K) anticoagulant vessels are widely used in basic research and clinical laboratories to collect, transport and preserve blood samples, which is not conducive to protecting specific molecular biomarkers such as RNA molecules in blood. In the process of sample preservation, if a sample is immediately frozen at − 20 °C or − 80 °C without any protective measures, subcellular structures are severely damaged, and the structure and activity of RNA can be altered, often irreversibly4. The quality of RNA in stored samples is directly affected by the method of sample storage5. In the case of improper storage, RNA in blood is rapidly degraded by RNase enzymes present in blood or the external environment. Previous research has shown that although EDTA-K2 can effectively prevent hemagglutination, it cannot maintain the stability of RNA in blood cells. Following cryopreservation, RNA is degraded by about 80% in a short time6. Therefore, the present work mainly compared the stability of messenger RNAs (mRNAs) and some non-coding RNAs (ncRNAs) in blood, explored the influence of relevant experimental operations on RNA degradation before and after RNA extraction, and quantitatively analysed on mRNA degradation in specimens and during experimental processes.In clinical diagnostic laboratories, 60–70% of errors occur in the pre-processing process of specimen analysis, most of which are caused by specimen collection, specimen processing and specimen storage9. The miRNAs regulate gene expression post-transcriptionally by interacting with and down-regulating target mRNA molecules. They are relatively stable and widely expressed in cells, tissues and organs throughout the body. In serum, plasma, saliva, urine, emulsions and other body fluids, miRNAs are highly stable extracellularly12. The miRNAs are associated with a variety of diseases and have been detected under a variety of pathological conditions13. CircRNAs are a common type of non-coding RNA produced by reverse splicing. Reverse splicing is an unconventional splicing event characterized by non-coding RNA molecules with circular structure formed by covalent bonds without a 5′-capped structure or a 3′-terminated polyadenylated tail. Since circRNAs are insensitive to nuclease, they are more stable than linear RNAs, but the mechanism by which cells eventually degrade circRNAs is still largely unknown compared to the mechanism of circRNA biogenesis15. LncRNAs are non-coding RNAs with a length of more than 200 nucleotides. Studies have confirmed the existence of hundreds of lncRNAs in serum/plasma. These long-chain molecules are stable and abundant, easy to be quantitatively detected, and have significant disease specificity13.The miRNAs are short (~ 22 nucleotides), non-coding, RNA molecules that control diverse biological processes, including cell fate determination, cell proliferation, cell differentiation and cell death16. Due to the presence of nucleases in the blood, most researchers doubt that extracellular RNA can remain stable for long periods of time17. For example, the miRNAs are generally considered to have a long half-life (T1/2), and can be stably present in serum and not easily degraded. Like other RNAs, miRNAs have a half-life, but exact data is rare in the literature19. The regulation of miRNAs is critical to the definition of cell identity and behaviour in normal physiology and disease. However, the dynamics of miRNA degradation and the mechanisms involved remain largely obscure, particularly in higher organisms20.Although great progress has been made on studying ncRNAs biosynthesis and functions, there are few reports on their degradation1/2) is important for understanding the regulatory mechanisms of gene expression and environmental changes that alter gene transcription levels21. Each method for measuring RNA levels has its advantages and disadvantages. The half-life of mRNA in tissues is generally determined by in situ hybridisation, which is time-consuming and labour-intensive9. Plasma ncRNAs can be quantitatively detected by real-time fluorescence quantitative PCR and microarray. If higher sensitivity is required, real-time quantitative PCR is a good choice22. The copy number of molecules in samples can be quickly and accurately determined by real-time quantitative PCR. The relative initial concentration is calculated based on the cycle threshold (Ct) value, and the half-life is calculated by establishing the regression linear equation over time. Due to the sensitivity of the method, the half-life of an RNA can be established even when it is expressed at low levels.The determination of RNA half-life  and regression analysis35.Therefore, a fast and reliable RNA half-life measurement method was established in the present study, based on real-time fluorescence quantitative PCR and supplemented by microspectrophotometry, to compare the half-life length of mRNAs and ncRNAs. Human-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin genes were used as internal reference genes for mRNA stability since their expression levels in cells or copy number in the genome is constant, and is less affected by environmental factorsThe present work focused on exploring the degradation of RNA at room temperature, using the same method to study the half-life of different RNAs and the influencing factors, and compare the degradation kinetics and characteristics of different RNAs in whole blood. The overall aim was to establish the optimal experimental and storage conditions, and thereby ensure the accuracy of experimental results.Fresh anticoagulant whole blood was used as experimental samples. Next, genomic RNA was extracted using a high efficiency blood RNA extraction kit  according to the instructions supplied with the kit. After extraction, RNA concentration and purity were calculated using a microspectrophotometer  according to the absorbance at 260 and 280 nm (A260 and A280). Reverse transcription was carried out in a 20 µL system with primers and reverse transcriptase . A 1 µL sample of the reverse transcription product was used for real-time quantitative PCR, and each gene was amplified by PCR. Primer sequences are shown in Table Whole blood was diluted to a series of concentrations, RNA was extracted using a high efficiency total blood RNA extraction kit, and a linear relationship between RNA concentration and dilution ratio was observed. The experiment was repeated three times.Extracted RNA was diluted to a series of concentrations and concentration was measured with a microspectrophotometer, revealing a linear relationship between RNA concentration and dilution ratio. The experiment was repeated three times.Extracted RNA was reverse-transcribed into cDNA and diluted to a series of concentrations. The GAPDH gene was amplified by real-time quantitative PCR, and a linear relationship between its Ct value and dilution ratio was observed. The experiment was repeated three times.Three factors and three levels of orthogonal experimental methods were employed. The three factors were the storage time of fresh whole blood at room temperature, the storage time of RNA at room temperature, and the storage time of cDNA after reverse transcription at − 20 °C. The Ct values of internal reference genes were determined by real-time quantitative PCR and the results were analysed by multivariate ANOVA and regression analysis. The optimal combination was obtained according to the results of orthogonal experiments. Whole blood was diluted 1:2, 1:4, 1:8, 1:16 and 1:32 with normal saline. RNA extraction and other related experiments were carried out under optimal combination conditions. GAPDH, β-actin and U6 genes were amplified by real-time quantitative PCR.t = 2(Ct0−Ctt), the relative template concentration (Nt) was calculated from the Ct values . K derived from the linear regression equation between the natural logarithm of the concentrations (Nt) and the incubation time, is the slope of the equation. The half-life(T1/2) was obtained using the equation37:When whole blood samples were incubated at room temperature, the RNA concentration gradually decreased with time. The half-life was calculated using a microspectrophotometer and real-time fluorescence quantitative PCR. Firstly, according to the equation Nt) are 1.00, 0.81, 0.39, 0.23, 0.19, 0.07, respectively, and then the natural logarithm of the concentration (lnNt) is transformed as 0, − 0.21, − 0.95, − 1.46, − 1.64, − 2.59 respectively. The linear regression is analysed using lnNt against incubation time, and the regression equation (y = − 0.0423x + 0.1262) is obtained, the slope (k) = 0.0423. Therefore according to the formula T1/2 = ln2/|k|, the half-life (T1/2) is 16.4 h.For example, the degradation of RNA in blood samples: the samples are incubated for 0, 12, 24, 36, 48, 60 h in order (Times), and the corresponding Ct values are 22.04, 22.34, 23.41, 24.15, 24.40, 25.78, respectively, and the corresponding concentration  orthogonal experimental table of three factors and three levels, with each factor at three levels. The β-actin gene was amplified by real-time quantitative PCR to measure the Ct value. The results are shown in Table Nine experiments were performed according to the L94 orthogoDirect comparison of the nine experimental results in Table p-values of fresh whole blood storage time at room temperature, RNA storage time at room temperature, and cDNA storage time at − 20 °C, and the p-values of the three influencing factors were all less than 0.05, and less than 0.01 for two of the influencing factors. Thus, these three factors had significant effects on RNA degradation, and the differences were statistically significant. However, there were some differences in the degree of influence. According to the regression analysis coefficient results, the storage time of RNA at room temperature had the least influence on RNA degradation, while the storage time of fresh whole blood at room temperature had the largest influence on RNA degradation. The effects of the three factors on RNA degradation were ordered whole blood storage time at room temperature > cDNA storage time at − 20 °C > RNA storage time at room temperature.In the table comparing inter-subject effects, we compared the According to the results of orthogonal experiments, the optimal combination of factors in this experiment was A1B1C1 . Therefore, in the process of RNA extraction and reverse transcription from whole blood, the time should be shortened as soon as possible, otherwise the subsequent results will be affected to a certain extent. Furthermore, blood was diluted to a series of concentrations for RNA extraction, followed by the amplification of GAPDH, β-actin, and U6 genes, and Ct values were recorded, all under optimal conditions. Taking the natural logarithm of the relative template concentration as a linear relationship with the dilution ratio, the correlation coefficient R values were 0.95, 0.99 and 0.93, respectively, all greater than 0.9, showing a good linear relationship.For half-life calculation based on microspectrophotometry, fresh anticoagulant whole blood was placed at room temperature, RNA was extracted every 12 h, and the concentration was measured by microspectrophotometry. The results are shown in Fig. For half-life calculation based on the real-time fluorescence quantitative PCR method, the degradation data at room temperature are shown in Tables 39. Because incomplete or degraded RNA may interfere with the accuracy of RNA half-life detection, the length of the amplified fragment is important to determine the RNA half-life40. Regarding genes, we selected β-actin, GAPDH and other housekeeper genes as internal reference sequences. Since their expression levels in cells or copy number in the genome is constant, and is less affected by environmental factors, their quantitative results reflect the number of cells or genomes contained in the sample41.In this study, a fast and reliable RNA half-life measurement method was established, based on quantitative real-time fluorescence PCR and supplemented by microspectrophotometry. Quantitative real-time fluorescence PCR can quickly and accurately determine the copy number of molecules in samples. qPCR was employed to detect changes in RNA content after incubating samples for different time periods, and the half-lives of mRNA and ncRNAs were comparedIn order to avoid errors caused by manual operation and differences in reagents and instruments, we conducted quantitative performance evaluation of the methods involved in the experimental process before the experiment. And the correlation coefficient R values were greater than 0.95, with good reliability Fig. , and the42. The orthogonal experiment results showed that the greater the number of factors involved in the experiment, the greater the impact on RNA degradation, and the faster RNA degradation. Multivariate ANOVA in the orthogonal experiment showed that the p-values of the three influencing factors were all less than 0.05, and p-values for two of the influencing factors were less than 0.01 . First, RNA concentration was measured by microspectrophotometry, we found that RNA degradation was fastest in the first 24 h (the slope of the dotted line was the greatest), and the slope of the dotted line became more gentle after 24 h. This shows that RNA is particularly prone to degradation which is consistent with literature reports44. The linear equation was calculated and the half-life of whole blood RNA at room temperature was 14.4 h. Second, using real-time quantitative PCR, the relative concentration was calculated according to the formula after taking the median Ct value, linear regression analysis was performed on the natural logarithm of the concentration vs. time, and the correlation coefficients were all greater than 0.9 (Table 46. However, we found that there seems to be no relationship between the length of the amplified fragments and stability because even though the amplified fragments are similar in length, their half-lives are still very different (Tables When processing actual samples, especially during mass inspection, experimentalists often ignore the effects of intermediate processes from specimen collection to specimen treatment on RNA concentrationt Tables , 8.47.In this experiment, the half-life results obtained by real-time fluorescence quantitative PCR and microspectrophotometry were essentially similar, and mRNAs and ncRNAs were measured simultaneously on the same platform. Therefore, we concluded that the storage method for samples directly affected the quality of RNA in the stored samples. RNA in blood is easily degraded during improper storage, and RNA is rapidly degraded by RNases present in blood or in the external environment. Storage at room temperature has a greater impact on RNA degradation1. With the development of biotechnology, detection methods will become more and more sensitive, hence it is particularly important to reduce variation during pre-treatment of specimens48. Due to the poor stability and short half-life of RNA, it is of great significance to select appropriate and professional methods to preserve blood samples to ensure the accuracy and reliability of test results. Especially in clinical trials, the acquisition of high-quality experimental samples is very important, hence more attention should be paid to the impacts of specimen analysis and processing49.The mRNAs and ncRNAs studied in the present work were from blood samples. Most previous studies have focused on tissue samples, but blood samples have advantages including being clearer, non-invasive, convenient for sampling, and amenable to continuous detectionSupplementary Information.


Transport of Nutrients, Metabolites, and Ions linked to Bioenergetics: Relevance to Human Pathology” volume 2, includes 6 contributions among which are 4 original articles, 1 brief research report, and 1 review. The second volume of the Research Topic follows the main scope of the first volume, i.e., adding novel and relevant information on the role of membrane transporters in bioenergetics. In particular, both aspects of basic and applied research are dealt with.The Research Topic on “The fundamental role played by membrane transporters in life is unequivocally testified by the occurrence of several cell dysfunctions occurring in all living organisms when these proteins are mutated, de-regulated, misfunctional, and/or mislocalized. Indeed, membrane transporters ensure the maintenance of the homeorhesis, that is the proper flux of metabolites, ions, and waste compounds required for cell homeostasis. Furthermore, during evolution, the membrane transporters acquired also the ability to mediate the uptake and efflux of xenobiotics, including drugs. This feature attracted the attention of pharma companies that nowadays focus a large body of their work on studying membrane transporters as both targets and transporters of novel and repurposed drugs.Karahoda et al.Notwithstanding the well-assessed function(s) of these proteins in cell life, several gaps exist in the knowledge of functional, structural, and regulatory properties of membrane transporters. In this frame, the original articles of the Research Topic, volume 2, find a scientific placement. Regarding functional characterization, Albrecht’s group performed a study describing the role of membrane transporters critical for placenta homeostasis. The paper evaluated the expression changes occurring during the first weeks of pregnancy, upon trophoblast differentiation. Very interestingly, the study highlighted that the glucose transporter (SLC2A1), the amino acid transporter (SLC7A5/SLC3A2), the iron transporter (TfR1), and the cholesterol transporters , increase their expression during placenta formation. This finding is in perfect agreement with the increased need for nutrients in the fetus development; indeed, the transporters under investigation being out of balance triggers compromised fetal growth Wannowius et al. employing combined approaches of site-directed mutagenesis, in situ localization, and fluorescent analyses of Ca2+ fluxes.The second article on the Research Topic is from Geyer’s group and deals with the characterization of rare genetic variants of the transporter SLC10A7, a negative regulator of intracellular calcium signaling. Therefore, is not surprising that mutations occurring at the level of the human gene are causative of severe diseases linked with alteration of calcium flux in cells. The study functionally characterized six SLC7A10 variants Tripathi et al.The third original article is from Fredriksson’s group and deals with the regulatory role played by SLC38A10 in glutamate homeostasis. Indeed, glutamate concentration needs to be maintained constant in synaptic clefts for allowing proper neurotransmission. In this scenario, the plasma membrane transporter SLC38A10 is considered a transceptor allowing the regulation of glutamate sensing through mTORC1 signaling. The paper shows data deriving from combined approaches of transcriptomic, protein arrays, and metabolomic. Interestingly, alterations of SLC38A10 are linked with lowering of cholesterol content, involved in neurodegeneration Qu et al.The last original article on the Research Topic is from Qu’s group and sheds light on the structure/function relationship of another group of glutamate transporters, belonging to the SLC1 family. Indeed, this family includes five high-affinity glutamate transporters mainly located in the brain, where they are responsible for glutamate reuptake to reduce excitotoxicity in synaptic clefts. The article, by employing site-directed mutagenesis coupled with chemical targeting, demonstrated that when EAAT2 interacts with substrate, the TM3-TM4 loop undergoes a proximal conformational change to TM7 allowing the transport cycle to occur with the elevator mechanism typical of this transporter family Berghaus et al.The brief research report from Weide’s group deals with the identification of the protein Pals1 as responsible for a key process in kidney homeostasis. Indeed, Pals1 is a member of the Crumbs protein complex that regulates cell polarization and junction formation. The transcriptome of Pals1-mice knockout highlighted the strong deregulation of several members of the SLC superfamily. The most affected ones are key players in kidney physiology, i.e., glucose transporter (SLC2 and SLC5), the sodium- and chloride-dependent neurotransmitter transporter family (SLC6 group), the amino acids transporters (SLC7 group), monocarboxylate transporters (SLC16 group), and organic cationic, and zwitterions transporters (SLC22 group). This has been linked with cystic formation typical of several kidney pathological conditions Drosophila melanogaster model which allows relatively fast and reproducible investigations. This is particularly relevant in drug discovery approaches considering that GABA transporters became hot targets for treating epilepsy, schizophrenia, and intellectual disabilities Fischer et al..The last article on the Research Topic is a review from Sucic’s group. The review is an interesting and comprehensive state of the art on the GABA transporters from the SLC6 family; indeed, the human genome encodes four isoforms of GABA transporters that are involved in modulating neurotransmission. The key relevant role of these proteins is demonstrated by the occurrence of several neurological disorders with a broad range of severity depending on the type of mutations. Over the years, a sizable number of mutations have been annotated on the genes encoding GABA transporters. The effect of single mutants has been studied using the in vitro, in vivo, and artificial intelligence tools represent a mandatory step forward to make a decisive increase in our knowledge of membrane transport phenomena.As a concluding remark of this volume 2, it can be highlighted that the Research Topic added further important novelties to the very broad and still incomplete field of membrane transporters. Notwithstanding, the full picture is still a low-resolution image, and its improvement represents a long-term challenge that requires substantial efforts in terms of experimental approaches and Research Topic and analysis of data. In this respect, multidisciplinary platforms including in silico,


We also demonstrated the pH-dependent selectivity of TiO2 microspheres towards different sizes of RNA. We were able to regulate the size range of extracted RNAs with simple adjustments in binding conditions used during the solid-phase extraction.While microRNAs are considered as excellent biomarkers of various diseases, there are still several remaining challenges regarding their isolation. In this study, we aimed to design a novel RNA isolation method that would help to overcome those challenges. Therefore, we present a novel phenol/chloroform-free, low-cost method for miRNA extraction. Within this method, RNA is extracted from cell lysate with an isopropanol/water/NaCl system, followed by solid-phase extraction using TiO MicroRNAs (miRNAs) are endogenous non-coding RNA molecules 19–23 nucleotides in length that are important for the regulation of gene expression and various cellular processes ,5,6. HowIn the last few decades, many methods for miRNA isolation have been developed from conventional DNA and RNA extraction methods, including phenol/chloroform-based extraction  or silica-based solid-phase extraction  . Commerc2—a cheap and easily available material in various commercial preparations often adopted for chromatography purposes. In proteomics, TiO2 materials are routinely used for selective enrichment of phosphorylated peptides due to the strong interactions between the TiO2 surface and phosphate groups of phosphopeptides in harsh acidic environments. Our previous results demonstrated that the TiO2 form, structure, and surface decoration can also significantly affect the affinity and selectivity for phosphorylated molecules [2 surface was used for effective interaction with DNA under strong acidic conditions (pH 2) as shown by Amano et al. [2-based miRNA isolation processes described by Jimenez et al. made use of self-prepared TiO2 nanofibers and only high concentrations of strong chaotropic salts (similar to SiO2 isolation protocols) without a low pH buffer system. This study demonstrated miRNA isolation from serum or cell lysate spiked with cel-mir-54 standard with 18.0% miRNA recovery. However, the described protocol was not size-selective, and a broad size range of RNA molecules under 500 bases was isolated [2 material. Currently, new approaches using various micro- and nanomaterials are being investigated with the aim of improving the yields and purity of extracted miRNA. Such materials offer unique characteristics, e.g., a high surface-to-volume ratio and useful magnetic, optical, or biological properties. Several studies have reported advanced nanomaterials suitable for both DNA and RNA isolation, such as graphene oxide nanoplatelets, zinc oxide nanowires and nanotubes, carbon nanotubes, and magnetite nanoparticles ,17,18,19olecules . Similaro et al. . On the isolated . These fIn our work, we aimed to develop an accessible and highly selective method for short RNA isolation (miRNA) and to avoid the extraction step with toxic phenol and chloroform.2-based miRNA isolation consists of a liquid–liquid extraction method using isopropanol/water/NaCl, followed by solid-phase extraction with porous 10 µm TiO2 microspheres under low pH in acetate, MES, or glycine buffer. The final elution of isolated miRNA is performed with Na2HPO4, which is compatible with PCR. The scheme of the experimental procedure, including specifically optimized conditions, is illustrated in Our protocol for TiO2 material; however, the contamination with RNAs of various sizes was observed. All protocol steps were then optimized to increase the purity of the isolated miRNA. First, we prepared a model RNA mixture consisting of genomic RNA (isolated from Jurkat cells with TRIzol Reagent) and oligo RNA standard hsa-miR-18a-3p. This mixture—containing 5.28 µg of pre-isolated genomic RNA and 0.55 µg of oligo RNA (23-mer)—was then used to optimize the binding conditions. Several washing and binding buffers within the pH range of 2.4–6 were tested for this purpose. 2-based short RNA isolation procedures under four distinct binding conditions differing in washing and binding buffer composition tested in triplicate. The corresponding data are summarized in 2 microspheres for short RNAs strongly depends on the pH of the selected buffer. The highest affinity of RNA molecules to TiO2 microspheres is present under strong acidic conditions  of citric acid in the washing and binding buffer which significantly increased the selectivity of TiOnditions . 2-based miRNA isolation from cells are schematically shown in To accomplish the isolation of miRNA from highly complex samples such as cells, the unique phenol/chloroform-free lysis and extraction protocol was developed. All steps prior to the TiO2 microspheres for further short RNA purification and isolation applying the general isolation protocol with the acetate buffer system. The amount of oligo RNA standard and sample composition during the whole isolation process was monitored by urea-PAGE. The results of the urea-PAGE analysis of all fractions collected from the whole isolation procedure are shown in 2-based isolation during the second part of the protocol. The residual proteins are visible in the supernatant fractions after TiO2 miRNA purification (lanes 3 and 7), whereas no proteins were detected in the elution fractions (lanes 4 and 8).The lysate was centrifuged  and A549 cells (one sample) and compared to RNA isolation using TRIzol Reagent. Elution fractions were analyzed by RT-qPCR in triplicate, and the results are summarized in 2-based method provides comparable yields to the commercial TRIzol Reagent isolation method, which is demonstrated by similar Ct values of RT-qPCR analysis. This also confirms the compatibility of the whole protocol, and mainly Na2HPO4 used for the elution of short RNA from TiO2 microspheres, with RT-PCR. To further confirm the feasibility of this new protocol, we performed the isolation of hsa-miR-18a-3p from two types of cell lines without any added standards. Hence, we show the ability of our protocol to analyze naturally occurring miRNA in cells. The TiO2 microspheres for short RNA isolation. They showed that the selective isolation of short RNA molecules is pH-dependent and the selectivity of miRNA isolation can be effectively modulated by changing the binding conditions. The isolation principle probably lies in the interaction of phosphate moieties of RNA with the TiO2 surface under an acidic environment. This strong interaction in combination with the porous structure of TiO2 particles  allowed selective binding of short RNA molecules, most likely due to steric hindrance of particle pores.Our results from experiments using model RNA mixture shown in 2 toward biomolecules containing negatively charged phosphate moieties can be modulated by hydroxy acids used as excluders, which allows us to further improve selectivity. This principle was also described for the extraction of proteins containing phosphate functional groups [2 more strongly than other tested carboxylic acids. For applications where a high selectivity for short miRNAs is not required, the relative yields of miRNA isolation can be increased up to 41% just by omitting the citric acid from the washing and binding buffer.Moreover, the selectivity of TiOl groups . In our 2-based miRNA isolation. For this purpose, the acetate buffer system proved to be more robust and easier to work with than the MES buffer. The protocol was also extended for the LLE method using an isopropanol/water/NaCl system  were tested for optimal protein removal and miRNA isolation, including acetonitrile (ACN), isopropanol, acetone, ethyl acetate, and n-hexane. When using immiscible or partially miscible solvents, RNA purification from proteins was poor . Isopropanol was selected as the best solution because its application together with NaCl resulted in almost complete phase separation. This was in direct contrast with the use of acetone or ACN, which stayed partially mixed within the aqueous solution after centrifugation. The amount of NaCl is also important for a proper phase separation, because at lower concentrations (less than 4 M), the phase separation was incomplete.2-based RNA isolation with a glycine buffer system. For effective cell lysis and LLE of RNA, it is necessary to keep the pH of the lysis solution approximately neutral, which prevents the loss of RNA molecules within the lysis procedure and exclusion from the aqueous phase into the interphase during the process of phase separation within LLE.For the isolation of a wider RNA size range with a higher yield, it is also possible to use a glycine buffer system. The pH of the lysis solution has to be adjusted to 6.4 using NaOH solution. After LLE, the pH of the acquired aqueous phase is lowered to ~2.4 by adding HCl before continuing with the general protocol of TiO2 microparticles can be applied for routine miRNA extraction from eukaryotic cells with excellent purity of the final product. This protocol could also be used for the simple separation of short RNAs from a mixture of RNAs differing in chain length, e.g., from pre-isolated genomic RNA. Glycine and acetate buffers are ideal for short RNA isolation from complex mixtures such as whole cell lysates. TiO2 microspheres in the environment of an acetate buffer with pH 4.4 possess high selectivity towards very short RNA molecules, resulting in extraordinary purity of miRNA isolated from such a highly complex mixture, which is clearly presented  by GL Sciences , were used as a carrier for the solid-phase extraction.TiOOligo RNA standard (23 nucleotides long) was designed analogically to the sequence of hsa-miR-18a-3p . It was used for spiking in the samples and to prepare model RNA mixture containing 5.28 µg of pre-isolated genomic RNA and 0.55 µg of oligo RNA (23-mer).6 cells was repeatedly frozen in liquid nitrogen and stored at −80 °C. RNA for model genomic RNA was isolated from Jurkat cells with TRIZol Reagent . Jurkat cells  were used for optimization of miRNA isolation from cells as well as for the preparation of a model genomic RNA. Jurkat cells cultivated in RPMI 1640 medium were harvested and washed with PBS, and a dry pellet containing 5 × 10A549 cells  were cultivated in MEM medium and then washed with PBS, and a dry pellet was repeatedly frozen in liquid nitrogen and stored at −80 °C.2 microspheres was dispersed in 80% ACN/0.1% trifluoroacetic acid (TFA). The suspension was centrifuged at 595× g for 1 min to separate the solid phase from the solution after each step of sample isolation. The microspheres were washed twice with suitable washing and binding buffer to remove the ACN/TFA solution. Then, 200 µL of the initial sample in the washing and binding buffer was added and the mixture was incubated for 60 min at 37 °C. The supernatant was collected for further analysis. Weakly and non-specifically bound compounds were washed out five times with 500 µL of the washing and binding buffer (repeating each step for 2 min at 37 °C). The elution of the adsorbed miRNA was performed with 100 µL of 200 mM Na2HPO4 for 10 min at 37 °C. The elution fraction was neutralized with 5% TFA to obtain an approximately neutral pH. The general isolation procedure described here was applied in all isolation experiments. First, 1 mg of TiO6) were lysed with 260 µL of lysis solution containing 100 mM sodium acetate, 2% Triton X-100, and 0.1% diethyl pyrocarbonate (DEPC) for 10 min. The sample was spiked with 1.10 µg of oligo RNA standard. The amount of RNA standard in the fractions was monitored by urea-PAGE throughout the protocol. The lysate was centrifuged at 10,000× g for 10 min. An aliquot of 30 µL was recovered for further analysis, and the remaining volume (230 µL) of the cell lysate was transferred into a tube containing the same volume (230 µL) of isopropanol with 53.8 mg of NaCl. The mixture was gently rotated for 10 min to gradually dissolve NaCl in acetate buffer. This led to the protein precipitation and separation of phases, where the top phase was organic and the bottom phase was aqueous. The proteins originating from the cell lysate were concentrated in the interphase after short centrifugation at 595× g for 30 s. The top phase was discarded, and the bottom aqueous phase was carefully aspirated using a Hamilton syringe and transferred into a clean microtube. Acetic acid  was added to adjust the pH to ~4.4 (checked with a microelectrode-equipped pH meter). Next, 200 µL of the aqueous phase containing RNA was added to 1 mg of TiO2 microspheres for further short RNA purification and isolation applying the aforementioned general isolation protocol with the acetate buffer system.Cells  before continuing with the aforementioned general isolation protocol of TiOPolyacrylamide gel electrophoresis (PAGE) was used for the subsequent analysis of the isolated miRNAs. The aliquot of separated fractions was mixed 1:1 with a sample loading buffer (89 mM tris(hydroxymethyl)aminomethane, 89 mM boric acid, 2 mM EDTA, 7 M urea, 12% Ficoll, 0.01% xylene cyanole FF/bromphenol blue) and incubated for 4 min at 70 °C. All collected fractions were then loaded onto a 0.75 mm urea-polyacrylamide gel (20% gel with 8 M urea), and electrophoresis was performed with the Mini-Protean System  at a constant voltage of 180 V in a Tris-Borate-EDTA running buffer. The gels were stained with SYBR Green II RNA Gel Stain  according to the manufacturer’s instructions. Stained gels were analyzed using the ChemiDoc XRS + System , with subsequent data processing in ImageLab software .Laemmli-SDS-PAGE electrophoresis with protein visualization by a silver staining method was used for the detection of protein residue in the samples of purified RNA ,24. StaiElution fractions were analyzed by RT-qPCR  according to the manufacturer’s instructions. First, miRNA was reverse transcribed into cDNA using hsa-miR-18a-3p-specific RT primer. A TProfessional Basic Thermocycler  was used for cDNA synthesis with the following program: 30 min at 16 °C, 30 min at 42 °C, and 5 min at 85 °C. The cDNA transcript was then used for detection by real-time quantitative PCR with TaqMan miRNA assay. The reaction was carried out on RotorGene RG-3000A  using a real-time quantitative program: 10 min at 95 °C, then 15 s at 95 °C and 60 s at 60 °C for a total of 40 cycles.2-based RNA isolation works in four buffer systems with different pHs. Moreover, in two of these (acetate or MES buffer), we were able to efficiently separate short RNAs from long RNAs, while still acquiring high miRNA yields. It was also experimentally proven that this method is compatible with a subsequent RT-qPCR analysis and achieved similar yields of native miRNA to the commercial TRIzol Reagent. Compared to conventional methods based on phenol/chloroform extraction, the proposed method provides several advantages, such as low cost , more environmentally friendly (green) chemistry, and most importantly, higher yield of miRNAs after extraction from real biological samples. It can be applied for routine isolation and identification of miRNAs in their analysis as a new molecular marker. Compared to traditional methods, the design and parameters of this TiO2-based method are better suited for the requirements of clinical practice, with an option for easy automation and miniaturization.In conclusion, a new highly selective two-step method for miRNA extraction from cell lysates was developed without any use of toxic reagents. We showed that TiO


We also found that BC002811 upregulated MMP2 and MMP9 and promoted GC cell metastasis partially through downregulating PTEN expression. BC002811 may act as a molecular decoy for the transcription factor SOX2, thereby inhibiting the transcription of PTEN by blocking SOX2 binding to the PTEN promoter. Our study advances the understanding of the role of BC002811 in the pathogenesis of GC and provides new molecular targets for therapeutic intervention against GC metastasis.There is increasing evidence that long non-coding RNAs (lncRNAs) are involved in the pathogenesis and progression of gastric cancer (GC), however, the underlying mechanisms remain poorly understood. In this study, we identified lncRNA BC002811 as a critical regulator of GC development and progression. BC002811 was upregulated in GC tissues and cell lines, and that high expression of BC002811 was indicative of a reduction in overall survival of GC patients. Our research reveals that BC002811 promoted GC cell proliferation, migration, invasion, and inhibition of apoptosis  Gastric cancer (GC) is one of the most common human malignancies worldwide, with more than 950,000 new cases are diagnosed every year, particularly in East Asia and Eastern Europe Long non-coding RNAs (lncRNAs) are endogenous transcripts longer than 200 nucleotides that lack protein-coding capabilities By comparing tumors to matched adjacent non-tumorous tissues using lncRNA microarrays, we previously demonstrated that a number of lncRNAs are abnormally expressed in GC PTEN promoter.In this study, we found that BC002811 expression was upregulated in GC tissues and cell lines. High BC002811 expression correlated with poor prognosis in patients with GC. By altering the expression of BC002811 in GC cells, we demonstrated that BC002811 increased proliferation, reduced apoptosis and promoted metastasis. Specifically, we propose that the pro-metastatic behavior of BC002811 may be through inhibition of the transcription of PTEN, through disruption of the interplay between the transcription factor SOX2 and the In situ hybridization (ISH) was performed using a digoxigenin (DIG)-labeled lncRNA BC002811 probe  following the manufacturer's instructions. Deparaffinized, graded alcohol-treated tissue sections were digested with proteinase K (40 μg/ml) at 37°C for 20 min, fixed with paraformaldehyde , incubated with prehybridization solution , and then hybridized with the DIG-labeled probe for 24 h. After incubating with blocking solution , sections were incubated overnight (4°C) with an alkaline phosphatase (AP)-conjugated anti-DIG antibody  and stained with AP substrate NBT/BCIP  for 4 h followed by nuclear fast red counterstaining. Image J software was used for quantitative analysis of the hybridization signals of the GC TMAs. According to the description of Chen et al. Human GC and corresponding non-tumorous tissue microarrays (TMA) were purchased from Shanghai Outdo Biotech . in vivo was investigated using 4-6 weeks-old male BALB/c nude mice . The cell lines used for the in vivo experiments were labeled with luciferase. For the tumor formation assay, the left or right thigh of each mouse was injected with 5 × 106 cells. All tumor xenografts were weighed and fixed for immunohistochemical analysis. For the lung metastasis model, mice were injected with 2 × 106 cells through the tail vein. After intraperitoneal injected of 150 mg/kg of D-luciferin, each mouse was imaged using an IVIS® Lumina II imaging system . All animal experiments were approved by the Laboratory Animal Ethics Committee of Guangdong Medical University.The function of BC002811 in tumorigenesis and metastasis GC tissue sections were incubated with hydrogen peroxide (3%) for 10 min, heated to 100°C in citrate buffer for 8 min for antigen retrieval, and subsequently cooled to 25°C. Sections were incubated with goat serum  in a humidified environment to block non-specific binding. Subsequently, the sections were incubated overnight (4°C) with primary antibodies, incubated with secondary antibodies  in a humid chamber, developed with diaminobenzidine tetrahydrochloride (DAB), stained with hematoxylin, and imaged. The immunostaining of GC TMAs was quantified by multiplying the staining intensity by the percentage of area with positive staining 2 Profiler PCR Array [Human Tumor Metastasis (PAHS-028Z)] following the manufacturer's protocols . The Human Tumor Metastasis qPCR Array allows simultaneous analysis of expression profiling of 84 key genes known to be related to human tumor metastasis. For signal detection, an initial denaturetion step of 10 min at 95°C was followed by 40 cycles each at 95°C for 15 s and 60°C for 1 min using the ABI Prism® 7900HT Real-Time PCR System . The RNA levels were normalized to five housekeeping genes. Fold changes in mRNA abundance were calculated according to the 2-∆∆Ct method The qPCR for microarray assay was carried out using the RTin vitro with T7 RNA polymerase  and Biotin RNA Labeling Mix (Roche) were incubated with cell protein extracts to allow the formation of an RNA-protein binding mixture, which was then captured on streptavidin beads. After SDS-PAGE and silver staining of the washed beads, proteins differentially bound to RNA were identified by mass spectrometry, and selected specific proteins were detected by western blotting.The RNA pull-down protocol was as previously described RIP was carried out using a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit  following the manufacturer's protocol. Protein A/G magnetic beads resuspended in lysis buffer were coupled to anti-SOX2 antibody or normal rabbit IgG (negative control) by incubating at 4°C for 4 h. The magnetic beads with the coupled antibodies were then incubated with the lysis mixture , and the RNA was eluted, and RT-PCR was performed. The detection primers for BC002811 are shown in GC cells that had reached 60%-70% confluence on coverslips in 12-well plates were fixed with paraformaldehyde (4%) for 15 min, followed by quenching of autofluorescence three times with 0.1 M glycine for 10 min each, permeabilized with Triton X-100 , treated with acetic anhydride (0.25%) for 10 min, and hybridized with Cy3-labeled oligonucleotide probes in hybridization solution at 45°C for 16 h. GC cells were then blocked with BSA  for 1 h, incubated overnight (4 °C) with anti-SOX2 antibody , and with Cy3-labeled secondary antibody . The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) and the cells were imaged using a TCS SPE confocal laser-scanning microscope .The ChIP assay was carried out using the EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore) following the manufacturer's instructions. The cells were cross-linked with formaldehyde (1%) and quenched with 125 mM glycine. The cross-linked chromatin was then sheared into 200-1,000bp fragments and precipitated with antibodies against SOX2. Fragmented chromatin DNA (10%) was used as the input, and normal rabbit IgG was used as a negative control. Fold enrichment of DNA was determined by qPCR with PTEN promoter-specific primers .EMSA was conducted using a LightShift™ Chemiluminescent EMSA Kit . Nuclear proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagent . The nuclear proteins in the binding buffer were then incubated with or without excess unlabeled wild-type competitors, followed by the addition of the biotin-labeled probes. For the supershift reaction, before the addition of the probes, an antibody against SOX2 was added to the reaction mixture. All protein-DNA complexes were separated by electrophoresis on a non-denaturing polyacrylamide gel and transferred to a membrane. Finally, antigens bound to the membranes were visualized by chemiluminescent detection.Details regarding additional materials and methods used in this study are provided in the in situ hybridization (ISH) in 163 pairs of clinical GC samples and adjacent non-tumorous gastric tissues using tissue microarrays  targeting BC002811 were analyzed. MTS assays showed that cell proliferation was diminished in AGS and HGC-27 cells in which BC002811 had been knocked down  out of 84 key genes involved in human tumor metastasis. MMP2 and MMP9 are well-known tumor metastasis markers, while PTEN is a well-characterized tumor suppressor gene that plays a major role in the metastasis of GC To further investigate the genes that may be involved in the promotion of metastasis by BC002811 in GC, we used human tumor metastasis qPCR arrays. As shown in Fig. ® assays revealed that upregulation of PTEN suppressed the invasion and migration of AGS and HGC-27 cells, and that BC002811 overexpression reversed this effect  followed by immunofluorescence of SOX2 demonstrated co-localization of BC002811 with SOX2 in the nucleus of AGS and HGC-27 cells  can facilitate malignant cell progression and promote tumor growth, invasion, and metastasis owing to their ability to degrade type IV collagen in the extracellular matrix and basement membrane Immunoprecipitation and mass spectrometric analyses of BC002811-interacting proteins led us to focus on the transcription factor SOX2. SOX2 is a critical regulator of embryogenesis and plays a major role in cell proliferation, differentiation, and cell fate determination PTEN promoter; (4) BC002811 co-localized with SOX2 in the nucleus of GC cells, in keeping with previous reports that transcription and epigenetic regulation of genes can be influenced by nucleus-retained lncRNAs LncRNAs can play important roles in cancer initiation, progression, and metastasis through precise regulation of gene expression at the transcriptional and epigenetic levels by acting as scaffolds, guides, activators, or decoys for their interacting proteins, such as histone modifiers and transcription factors Decreased expression of PTEN has been reported to be involved in worse patient outcomes in GC PTEN promoter, which consequently leads to suppression of PTEN transcription. The decrease in the level of PTEN protein can further stimulate MMP2 and MMP9 expression, which ultimately promotes GC cell metastasis (Fig. In summary, we identified BC002811 as an oncogenic lncRNA involved in GC growth and metastasis. Our findings suggest that BC002811 binds to SOX2, thereby disrupting the interplay between SOX2 and the Supplementary materials and methods, figure and tables.Click here for additional data file.


Caenorhabditis elegans and humans, i.e. that the novel NMD intermediate occurs after a prior ribosome elicits NMD. Our genetic analysis of C. elegans’ SMG-6 supports a central role for SMG-6 in metazoan NMD, and provides a context for evaluating its function in other metazoans.Nonsense-mediated mRNA decay (NMD) protects cells from the toxic and potentially dominant effects of truncated proteins. Targeting of mRNAs with early stop codons is mediated by the ribosome and spatiotemporally aligned with translation termination. Previously we identified a novel NMD intermediate: ribosomes stalled on cleaved stop codons, raising the possibility that NMD begins even prior to ribosome removal from the stop codon. Here we show that this intermediate is the result of mRNA cleavage by the endonuclease SMG-6. Our work supports a model in which ribosomes stall secondary to SMG-6 mRNA cleavage in  Drosophila, mammals). Furthermore, the mRNA fragments made during NMD are rapidly cleared by downstream decay in vivo and are thus challenging to study.Nonsense-mediated decay (NMD) is a translational surveillance pathway where premature stop codon (PTC) containing mRNAs are recognized and degraded (reviewed in ). Targetskih-2 and pelo-1  selection nor ribosome subtraction was performed. 1ug of RNA was used as input for 3′ RACE library preparation. For ‘3′ OH + 3′ P 3′ RACE’, total RNA was first treated with T4 PNK (NEB) to remove 3′ phosphates. The T4 PNK reaction was cleaned up with phenol/chloroform extraction followed by RNA precipitation, and then used in ligation. For ‘3′ OH 3′ RACE’ libraries, total RNA was directly used in ligation. Volumes and conditions were per similar, published protocols . PreadenC. elegans genome  including annotated splice junctions using STAR v2.7.3a (unc-54(PTC) experiments, reads were mapped to a custom genome containing the introduced mutations at the unc-54 locus. This was done by masking the bases of the unc-54 locus and creating a separate chromosome and annotation file for the unc-54 locus. All downstream analyses were restricted to uniquely mapping reads. Normalized Read Density for metagene analyses was computed as previously described  and liquid nitrogen in a mortar and pestle. Ground powder was mixed with ice cold PLB and clarified via a 10’ spin at 10 000 rcf at 4°C. Ribosome/RNA was quantified with a nanodrop and OD260 units were used to calculate the amount of RNaseI to use . RNA was treated for 30 min at room temperature with RNaseI  and loaded onto a 10–50% sucrose gradient. Gradients were spun in an SW41 Ti rotor in an ultracentrifuge at 35 000 rpm for 4.5 h. Monosomes were collected on a fractionator and digested with proteinase K. Monosome RNA was cleaned up by acid phenol chloroform extraction, precipitated, and stored in TE 7.4. 2–3 ug of purified monosomal RNA was run on 15% polyacrylamide gel and size-selected for 15–18nt, 19–26nt or 28–30nt footprints. Gel-purified RNA was treated with T4 PNK (NEB) to remove 3′ phosphates. RNA was then extracted with phenol chloroform, precipitated, and resuspended in TE pH7.4. Preadenylated adaptor (/5rApp/NNNNNNNNAGATCGGAAGAGCACACGTCT/3ddC/) was ligated onto RNA 3′ ends with T4 RNA Ligase 2 truncated KQ (NEB). Adaptor-ligated RNA was run on a 10% polyacrylamide gel, size selected, and processed as per reverse transcriptase, circligase, and PCR in the 3′RACE protocol.Frozen animal pellets were ground with frozen PLB  and stored at −70°C were boiled in 1xSDS loading buffer . Samples were run on a 4–20% Mini-PROTEAN TGX Stain-Free Protein gel . Protein was transferred to a low background fluorescence PVDF membrane (Millipore). The membrane was blocked in 1% nonfat milk in 1× TBST. The anti-FLAG antibody (Sigma F1804) was used at a 1:1000 dilution to detect FLAG-tagged SMG-6 protein. Secondary antibody staining was performed with 1:15 000 LI-COR goat anti-mouse (LI-COR). Imaging was performed using a LI-COR Odyssey Imaging System (LI-COR) and quantification done in ImageJ.skih-2 pelo-1 double mutant.We set out to understand the nature of the nuclease that cleaves NMD targets by examining the chemistry of the resultant 3′ termini genome-wide. The chemistry of 3′ termini reports on the nuclease that made them: metal-independent nucleases usually leave a 2′–3′ cyclic phosphate (resolved to a 2′ P or 3′ P) or a 3′ P, while metal-dependent nucleases leave a 3′ OH . We madexbp-1 gene. Site-specific cleavage of xbp-1 mRNA by the metal-independent endonuclease IRE-1 generates a 2′–3′ cyclic phosphate end  important for NMD in humans and osophila , and SMGosophila ,32. We tt in NMD . Given tin vitro  (unc-54(r293) is an allele of unc-54 that encodes a functional UNC-54 myosin heavy chain protein required for animal movement, and its phenotype is uncoordinated because the unc-54(r293) mRNA is targeted by NMD. Animals with functional NMD are thus uncoordinated (Unc) and animals lacking NMD are not uncoordinated (non-Unc). A simple assay for Unc is to place the animals in a liquid and count the frequency with which they thrash. unc-54(r293) animals were non-Unc when crossed with each smg-6 mutation, indicating de-repression of unc-54(r293) and loss of a functional NMD pathway  ,34. unc-y Figure . Thus, esmg-6 exhibited maternal rescue. Maternal rescue is a phenomenon wherein homozygous mutant progeny fail to manifest a phenotype in the first generation. This can be due to cytoplasmic inheritance of functional wild-type protein or mRNA packaged from the mother into the developing egg. Subsequent generations exhibit the phenotype as the wild-type protein (SMG-6) is diluted out and lost. Maternal rescue has been documented before with prior smg-6 mutants (smg-6 knockdown at the mRNA level (see Discussion).In the course of the above experiments, we noticed that all the active site mutations of  mutants , and we  mutants . The matsmg-6. UNC-54 is expressed in the body wall muscle, and a requirement for smg-6 in other tissues could not be tested with the unc-54(r293) assay. Therefore, we turned to a recently published cell-specific NMD reporter  (encoding the SMG-1 kinase), smg-4(az152) (encoding UPF3), and smg-5(r860) (encoding SMG-5)  ,36,37. Ig SMG-5) . A smg-6m Figure . Thus wesmg-6 and the other smg factors in C. elegans, as loss of function of any one factor is sufficient to break the NMD pathway as assayed by genetic suppression of NMD reporters such as unc-54(r293) (unc-54(r293) and R2 reporters. To explore a requirement for the SMG-6 PIN domain in NMD more globally in C. elegans, we examined its role in repression of endogenous NMD targets via RNA-seq.One prominent model in the NMD field is that SMG-6 cleavage is one option of several degradation pathways by which NMD represses its mRNA targets . We note54r293) . Our gen93 . Our smg-6 and its PIN domain. To identify endogenous NMD targets, we first performed RNA-seq in a smg-1 mutant and examined upregulated mRNAs using DESeq2  as well as indirect targets (downstream consequences of de-repression of NMD substrates). For the sake of simplicity, we refer to both as targets; the fact that some are indirect does not invalidate the approach.NMD is known to be required for the repression of several endogenous mRNAs, providing a diverse array of NMD substrates by which we could test for a requirement for 2 Figure . SMG-1 i elegans . Using D elegans  38). Th. Thsmg-6smg-6 PIN targets by examining mRNA expression in the smg-6(D1070A) mutant. This approach identified 1255 smg-6 targets at an adjusted P-value cutoff of 0.01  the gene is uniquely regulated by one of smg-1 or smg-6 or (B) a failure to detect a statistically significant difference due to a relative increase in effect size in one set of libraries (e.g. due to deviations in fold change or dispersion). To discern between (A) and (B), we examined the smg-6 targets in the smg-1 mutant. Of the 108 smg-6-specific targets, 106 exhibited an increase in expression in the smg-1 mutant, falling just below the cutoff and thus not making the smg-1 target list  , with sosmg-6(D1070A) into the skih-2 pelo-1 strain and performed Ribo-seq. In the absence of active smg-6, stop codon cleavages were reduced genome-wide .We crossed an active site point mutation of smg-6D70A into smg-6 strain, we noticed that 21nt Ribo-seq reads at the stop codon for SKI/PELO targets did not differ with and without smg-6  in C. elegans  are degraded through mechanisms genetically distinct from smg-6 and NMD.15–18nt Ribo-seq reads persisted for several genes in the 9 Figure . The 15– elegans . We concOur work thus far supported the idea that SMG-6 cuts the mRNA, stalling ribosomes and giving rise to 15–18nt Ribo-seq reads at and around the PTC. We set out to understand the events immediately preceding ribosomal stalls at and around stop codons by considering two possible models for their generation.skih-2 and pelo-1-dependence of ribosome stalling . Th. ThC. elC. elegans and humans  occurring in both xbp-1), we knew that cleavages occurred at the stop codon, as well as at positions up- and downstream , we observed a sharp accumulation of 3′ ends upstream of the stop codon. We would not expect such 3′ ends under the first model of Figure Our metagene analysis Figure  demonstrm Figure –C. Consim Figure , a resulm Figure , B. Impom Figure . The 15–c27a12.4 Figure unc-54 locus (see below).In a few cases (e.g.unc-54(r308) allele, with additional stop codons near the expected stop codon. The reporters were made via CRISPR/Cas9 edits of the endogenous unc-54 locus. The reporters (unc-54(PTC1), unc-54(PTC2) and unc-54(PTC3)) encode identical ∼6200nt mRNAs except 1–2 nucleotides near the site of termination. Importantly, the mutations change the precise site of termination as well as the last few codons and amino acids.The above gene-specific differences in Ribo-seq reads could arise from differences in the sequence of the genes and/or from spatiotemporal differences in each gene's expression and regulation. To better understand the determinants of cleavage, we generated a series of three reporters Figure . The thrskih-2 pelo-1 background and performed 15–18nt and 28–30nt Ribo-seq. As expected, all three samples exhibited a high correlation of Ribo-seq read counts and distributions ). Interestingly, recent work has shown that some TUTases prefer 3′ Gs  strain we observed a large number of 15–18nt Ribo-seq reads downstream of the stop codon  reporter has a stop codon (TGAC) that confers high levels of readthrough across eukarya ). If the ribosome directed cleavages via its A-site , we would expect to capture such cleavages only when the trailing ribosome bypasses the unc-54(PTC2) stop codon. Under model two we would expect 15–18nt Ribo-seq reads to become less abundant further downstream of PTC2, as the chances of a ribosome encountering a 3′ end generated by SMG-6 and the exosome increase as the ribosome elongates downstream. Thus model two predicts an absence of reads at the next in-frame stop codon downstream of PTC2, as appears to be the case in our data.Interestingly, we did not observe reads at the next in-frame stop codon (∼100nts downstream of unc-54(PTC2) 15–18nt Ribo-seq read pattern is reminiscent of similar patterns observed on some endogenous NMD targets . Consistent with this logic, the earlier studies that observed mild effects of hSMG6  we observe directly overlap the stop codon, they represent an earlier intermediate on the NMD pathway than those reported previously  at the would-be cleavage sites. We also did not observe this  and scripts for this study are therein (220606_kimModenaSmg6Paper).Data is available through SRA via Bioproject number PRJNA819566. A list of all libraries generated in this study are in gkac681_Supplemental_FilesClick here for additional data file.


Our work suggests a functional and regulatory role of RNA editing in the pathogenesis of AML and we extended our analysis to gain insight into the factors influencing altered levels of editing.Several studies have documented aberrant RNA editing patterns across multiple tumors across large patient cohorts from The Cancer Genome Atlas (TCGA). However, studies on understanding the role of RNA editing in acute myeloid leukemia (AML) have been limited to smaller sample sizes. Using high throughput transcriptomic data from the TCGA, we demonstrated higher levels of editing as a predictor of poor outcome within the AML patient samples. Moreover, differential editing patterns were observed across individual AML genotypes. We also could demonstrate a negative association between the degree of editing and mRNA abundance for some transcripts, identifying the potential regulatory potential of RNA-editing in altering gene expression in AML. Further edQTL analysis suggests potential   •Increased levels of RNA editing are a predictor of poor survival in AMLs•Differential levels of RNA editing are observed among AML genotypes•mRNA abundance negatively correlates with the degree of editing of some transcripts•RNA editing variation is partly explained by cis-regulatory mechanisms Cancer systems biology; Cancer; Transcriptomics Editing of RNA, alternatively termed as “RNA-DNA differences” (and hereafter RDDs), is another such putative “epitranscriptomic” modification that may alter gene expression and cellular phenotype, without permanently altering the DNA sequence. Although not the only modification, the major editing event is the alteration of Adenosine (A) to Inosine (I), which in turn is read as Guanine (G) by the translational machinery. This activity is predominantly catalyzed by the ADAR enzymes.Acute Myeloid Leukemia (AML) is an aggressive, often fatal hematological malignancy, where the normally exquisitely regulated processes of self-renewal, proliferation, and differentiation within the hematopoeitic system are aberrantly coordinated.,,,,,,AZIN1 (S367G) in Hepatocellular carcinoma and RHOQ (N136S) in Colorectal cancer, have been shown to be associated with tumorigenesis. Similar trends are observed for global measures of RNA editing, where the majority of cancers have been reported to have elevated levels of editing. Furthermore, it has been shown for most tumors that increased levels of editing are associated with poor survival outcomes.,Although RNA editing was originally described over 30 years ago, only recently, with the advent of deep sequencing technologies, have millions of RDDs been documented across healthy individuals and several cancers.PTPN6 (SHP1) transcript was linked to disease progression.,,,In an earlier study across AML patient samples, a hyper-edited event at an intronic branchpoint of the HAUS3, PUS1, SDHAF2, SRP9, and ZNHIT3 were determined to be predicted vulnerabilities in AML by our previous genome-wide CRISPR -dropout screen in AML cell lines.A schematic of the analytic pipeline for the study is shown in RNAEditingIndexer tool,,,We subsequently used two approaches to quantify the degree of RNA editing to help understand its role in AML. Using the ,It has been widely reported that RNA editing is altered in several cancers.Within the TCGA cohort, we further aimed to look at how RNA editing differs across different AML mutational genotypes. We hypothesized that the heterogeneity in RNA editing levels in AML may relate, at least in part, to the genetic heterogeneity evident within it. As previously documented, we observed genetic heterogeneity within our cohort and used an up-to-date disease classifier to subdivide AML into pathogenetic subtypes.th percentile were considered to be hypo-edited . Sample classification, including clinical data, is shown in We next sought to determine any link between RNA editing and survival, and, utilizing similar methodologies to previous studies D, we claTo exclude an association of AEI with other prognostic variables within the TCGA data, we therefore extended this analysis by fitting a multivariate model using several clinical characteristics of known prognostic significance, such as mutation status, age and sex as additional variables.SOCS2-AS1). It has recently been reported that SOCS2-AS1 modulates FLT3-ITD activity via sponging miRNA-221 activity to maintain STAT5 signal transduction.FLT3 mutational status between the number of patients with  and those that lacked an RDD . Moreover, the AEI also did not alter between patients that carried or lacked the FLT3-ITD mutation . At FDR 20% , fifteen editing events, predominantly located in non-protein-coding DNA, conferred a markedly poorer survival outcome for those patients who either demonstrated or lacked an editing event A. The momutation C. Multivn status F. HoweveSOCS2-AS1 in comparison to non-edited patients (N = 119)  D. We nexbundance E. Taken SOCS2-AS1 expression levels, we next analyzed whether this was also a general phenomenon across all RDDs  we found 966 sites  corresponding to 610 genes , where the degree of editing significantly correlated with the expression level of their transcript B. Remarkpt level B. We havA levels .To understand the functional relevance of the genes that are associated with rRDDs, we then sought to estimate the extent of their overlap with genes differentially expressed between blasts from patients with AML and from healthy individuals (see methods), to determine if this difference might relate to their edited status. Of note, we found that 33 of 337 genes  linked to rRDD, overlapped with genes up-regulated in the BeatAML dataset , but tha,Quantitative trait loci (QTLs)-mapping is an approach that has been widely used to find an association between genetic variants and phenotypic variation, such as gene expression (eQTL),2 > 0.8) of the genomic variants was taken into account, we found only five genes   analysis from the same dataset and overlapped variants of interest with our edQTLs identified above. As reported in several studies, we performed eQTL association analyses with the same imputed genotypes within a 1 MB window from the TSS of the genes that are expressed in all the samples . At FDR 10% (2.87e-04), we found 3274 genes to have a significant association with a nearby genomic variant. However, when the linkage disequilibrium (LD) structure (Rd SENP5)  to have Upon testing 3590 RDDs, we found 200 edQTLs to have an association with the nearby genetic variant (edSNPs). To determine the potential functional relevance of these associations, we sought to determine the extent of overlap of the rRDD. We found 27 (13.5%) of the 200 edQTLs  to overl,,https://forge2.altiusinstitute.org/) which detects tissue and cell-type specific enrichment in epigenomic regions including DNase I hotspots, histone mark broadPeaks and Hidden Markov Model (HMM) chromatin states.https://screen.encodeproject.org/) integrated DHS regions with multiple epigenetic signals, namely H3K4me3, H3K27ac, and CTCF ChIP-Seq and defined candidate Cis-Regulatory elements (cCREs) across different cell lines.We noted that the majority of edSNPs are located in non-protein-coding DNA B. Multip,,A-to-I RNA editing is a post transcriptional modification that is ubiquitous across Metazoans.More recently, global measures of RNA editing were estimated in patient cohorts from the TCGA and demonstrated elevated levels of editing across multiple primary solid-organ cancers. These differences in AEI are largely explained by ADAR1 expression levels.CEBPA, and NPM1 mutations, appear to have a lower degree of RNA editing in comparison with patients carrying high-risk mutations (i.e. patients with mutated chromatin regulators and TP53). The AEI index is positively correlated with ADAR1; however, we observed no significant differences in ADAR1 expression levels across the AML genotype . A recent study reported that ADAR2 is significantly downregulated in patients with t or Inv16 mutations, carrying RUNX1-ETO and CBFβ-MYH11 fusion proteins. Tenen and colleagues have experimentally demonstrated RUNX1-ETO repression of ADAR2 expression and proposed a mechanism whereby hypo-editing of specific transcripts by ADAR2 was implicated in the pathogenesis of t AMLs.We further observed significant differences in the editing levels across patient samples with different AML genotypes. Of note, in general, the genotype groups with low risk, including good risk translocations, ,Previous studies in breast invasive carcinoma (BRCA), liver hepatocellular carcinoma (LIHC), head and neck squamous cell carcinoma (HNSC) and glioblastoma multiforme (GBM), have demonstrated that the AEI correlates with overall survivalSOCS2-AS1  predicted a poor outcome. In addition to RDD at the SOCS2-AS1 locus, we found many sites that demonstrated an association between the degree of editing and steady-state mRNA levels. However, this relationship appears complex given that the presence of an editing event was often associated with a higher steady state mRNA for the primary transcript, but the degree of editing inversely correlated with the mRNA abundance. Although initially counterintuitive, this suggests that RNA editing may be an additional layer of control to regulate transcript abundance, and perhaps particularly in some highly abundant transcripts, although whether this editing alters RNA stability or turnover remain mechanistically elusive and warrants further investigation. Moreover, the significant overlap of these genes with those overexpressed in AML vs healthy controls suggests that, along with several genetic and epigenetic factors, RNA editing in AML may contribute to disordered transcription and the malignant phenotype.We further assessed the potential effects of each individual editing site on the patient’s survival outcome. Interestingly, we found that the presence of an RDD in the exonic location of the ncRNA Cis-regulatory variants. Earlier studies have shown that several RNA binding proteins (RBPs) facilitate editing by interacting with ADARs or through binding to Alu elements in a tissue-specific manner.In summary, we present the first large-scale RNA editing analysis from AML patient samples. Our results demonstrate that increased levels of editing are associated with poor prognosis, that the AEI varies across AML genotypes and that there is an association between the degree of RNA editing and expression levels of multiple genes, some of which are dysregulated in AML in comparison to normal hematopoiesis . Our edQTL analysis revealed that one of the factors potentially influencing the altered levels of editing are Our study aims to understand the potential role of RNA editing in the pathogenesis of AML; however, it has some limitations. To enable us to study RNA Editing, we have accessed the most comprehensive transcriptomic data of patients with AML from the TCGA, a dataset that also benefits from having cytogenetic data regarding mutational events and clinical outcome data, allowing us to further correlate RNA editing with these disease characteristics. However, an obvious drawback of this data is that it lacks a matched control dataset, both at the level of the individual patient and an appropriate cell type. Therefore, to study the global differences between AML and healthy controls we utilized transcriptomic data from a validating external dataset, the BeatAML dataset. Within the BeatAML cohort, we validated our findings by observing AMLs to have an elevated degree of RNA editing in comparison to healthy controls.fourteen individual sub-groups. Given our relatively small dataset of 151 patients, to allow us any statistical power, we therefore had to rationalize this classification system, collapsing similar biological and clinical groups together into a more manageable seven genetically similar groups. Decades of genetic research have taught us that increasing the sample size can add significant mechanistic granularity, therefore our underpowered study may have underestimated or missed associations between RNA editing and AML biology. However, despite our limited cohort we were still able to (i) demonstrate variable RNA editing across AML genotypes, (ii) describe that higher levels of editing are a predictor of poor outcome, (iii) observed elevated degree of editing in AMLs in comparison to healthy individuals, (iv) demonstrate that RNA editing is one of the contributors to altered gene expression in AML and (v) have identified edQTL to identify putative cis-regulatory regions that regulate RNA editing, and could validate the clinically relevant findings i-iii) in an independent AML cohort, the BeatAML dataset.Although our cohort is the largest with transcriptome, genome, and clinical data available, another limitation of our study is the cohort sample size, which is relatively modest at 151 patients, with another 135 in the validating dataset. However, AML is a clinically heterogeneous disease that is characterized by a large number of recurrent gene mutations and chromosomal abnormalities. A recent panel-based genome study, performed on 1540 patients, classified AML into em540@cam.ac.uk).Further information and requests should be directed to the lead contact, Eshwar Meduri . With the trade-off between the sequencing costs and the number of reads, it is not feasible to capture editing levels of all Adenosines in large cohorts. To overcome this, a metric called the Alu Editing Index (AEI) was designed to measure global editing levels in each sample.R172 mutations sub groups contained only one patient, that was excluded, whereas patients with NPM1, mutated chromatin+TP53, good risk translocations+CEBPA, > 2 genomic subgroup eligibility and no class defining lesions+driver mutations, were present at a range of 6–34 patients . Regulatory editing events are quantified by overlapping filtered significant events fulfilling both the above criteria. As an exemplar, we chose RDD at chr12: 935429492 in AS1 gene A.https://portal.gdc.cancer.gov/) and was processed using Affymetrix library files and converted to the VCF format using https://github.com/freeseek/gtc2vcf. Prior to imputation we followed several QC steps as follows 1) we filtered for common variants (MAF > 0.01) and deviations from HWE (1e-05). 2) Two samples were removed after checking for high levels of missing data. Data was pruned at LD > 0.2. 3) We excluded high LD and non-autosomal regions from the pruned file. 4) Related individuals  were further discarded.All the patient samples were genotyped on the Affymetrix Genome-wide Human SNP Array 6.0. Raw data was downloaded from GDC data portal  and the imputed genotypes were performed using QTLTools package.As described in the GTEx consortium eQTL analysis pipeline, read counts were normalised using trimmed mean of M values (TMM).FORGE2 command line analysis was performed on 4,305 top edSNPs using standard settings and ENCODE data (1000 background repetitions, ENCODE DNase I hotspot data analysis setting.qvalue package was utilized to adjust the p values during multiple testing. Unless otherwise stated, all the statistical tests were considered to be significant with a p value < 0.05. All the statistical tests were performed using the R package.We used the Wilcoxon test for comparisons between two groups and the Kruskal–Wallis test for comparisons between more than two groups. To compare two continuous variables we used Pearsons correlation method but with additional covariates we applied multiple regression model. Kaplan-Meier analysis was used to estimate survival outcomes and hazard ratios were calculated using a multivariate Cox proportional hazards regression model. The phs000178.v11.p8 and BeatAML: phs001657.v2.p1. All the data generated is available along with this article.The data used for the analyses described in this manuscript were obtained from the dbGaP website under accession number TCGA: