Further, its role in NPC cell growth and metastasis was verified in vitro and in vivo. The researchers conducted the RNA pull-down assays, mass spectrometry (MS), dual-luciferase reporter assays, RNA immunoprecipitation (RIP) assays, and the MS2-RIP assays were then used to identify the lnc-MRPL39-2:1-interacting proteins and miRNAs. We found that lnc-MRPL39-2:1, which was highly expressed in in NPC tissues, was related to a poor prognosis in NPC patients. Furthermore, lnc-MRPL39-2:1 was shown to induce the growth and invasion of NPC by interacting directly with the Hu-antigen R (HuR) to upregulate β-catenin expression both in vivo and in vitro. Lnc-MRPL39-2:1 expression was also suppressed by microRNA (miR)-329. Thus, these findings indicate that lnc-MRPL39-2:1 is essential in NPC tumorigenesis and metastasis and highlight its potential as a prognostic marker and therapeutic target for NPC.Long non-coding RNAs (lncRNAs) have been to regulate tumor progression and therapy resistance through various molecular mechanisms. In this study, we investigated the role of lncRNAs in nasopharyngeal carcinoma (NPC) and the underlying mechanism. Using lncRNA arrays to analyze the lncRNA profiles of the NPC and para-tumor tissues, we detected the novel lnc-MRPL39-2:1, which was validated by  Nasopharyngeal carcinoma (NPC) is a type of head and neck cancer that originates in the nasopharynx and displays a high metastatic potential Long non-coding RNAs (lncRNAs) are a group of endogenous transcripts that are longer than 200 nucleotides (nt) in length and are not translated into proteins Figs. S1). The top 35 significantly-altered lncRNAs are shown in Fig. . A few of the differentially expressed lncRNAs (four upregulated and three downregulated) were selected for further analysis . As one of the most highly upregulated lncRNAs detected, we focused primarily on lnc-MRPL39-2:1. To explore the role of the lnc-MRPL39-2:1 in NPC progression, we analyzed the differential expression of lnc-MRPL39-2:1 in the NPC tissues and the non-tumor tissues in the Gene Expression Omnibus database . The results indicated that lnc-MRPL39-2:1 was expressed at a high level in NPC tumors compared to non-tumor tissues . Using quantitative PCR (qPCR) and the RNA-fluorescence In Situ Hybridization (RNA-FISH) techniques for validation, we confirmed lnc-MRPL39-2:1 was significantly upregulated in NPC tumor tissues compared with non-tumor tissues. Moreover, we established a direct link between lnc-MRPL39-2:1 expression levels and the advanced clinical stages of NPC patients . ISH arrays were used to further identify the expression and localization of the lnc-MRPL39-2:1 in NPC. Lnc-MRPL39-2:1 expression was negligible in nasopharyngitis tissues, whereas upregulated lnc-MRPL39-2:1 expression was detected in 75.4% of the NPC samples (92/122) had a higher lnc-MRPL39-2:1 expression . The expression levels of the lnc-MRPL39-2:1 were dependent on many factors, including the NPC patients' tumor (T) stage, nodes (N) stage, and the tumor-node-metastasis (TNM) stage. However, it was not related to other pathological characteristics such as sex, age, and local treatment failure of the patients (Table S1). Survival analysis indicated that upregulated lnc-MRPL39-2:1 expression was associated with a poor overall survival (OS) rate and progression-free survival (PFS) . Multivariate analysis of the independent prognostic factors for OS and PFS indicated a negative correlation of lnc-MRPL39-2:1 expression and TNM stage with the prognostic factors for the OS and PFS (Table S2). Subsequent survival analysis based on a combination of the lnc-MRPL39-2:1 signature and TNM stage showed significant differences in the OS and PFS values amongst the low- (low lnc-MRPL39-2:1 expression and early TNM stage), intermediate- (high lnc-MRPL39-2:1 expression or advanced TNM stage), and high- (high lnc-MRPL39-2:1 expression and advanced TNM stage) risk groups, thereby indicating that a combination of the lnc-MRPL39-2:1 expression and TNM stage could act as a potential marker to predict the NPC prognosis . In conclusion, the data revealed that lnc-MRPL39-2:1 expression was increased in NPC and was related to the poor prognosis.To determine the lncRNAs that play important roles in the NPC progression, we analyzed the expression profiles of a panel of lncRNA in the five selected pairs of NPC and para-tumor tissues using lncRNA arrays. The results indicated the presence of 262 dysregulated lncRNAs, a high proportion (48.15%) of which were intergenic . The lncRNAs ranged from 400 bp to 800 bp in size with a range of classifications, and chromosomal locations . Furthermore, lnc-MRPL39-2:1 expression was primarily localized in the cytoplasm of the NPC cells . The complete lnc-MRPL39-2:1 sequence was then determined by the 5' and 3' cDNA ends (RACE) assay . Searches of the phylogenetic codon substitution frequency (PhyloCSF), CPAT, and LNCipedia databases, confirmed the absence of protein-coding potential; however, RegRNA 2.0 analysis revealed that the lnc-MRPL39-2:1 transcript contains two open reading frames (ORFs). To determine translation potential of these two lnc-MRPL39-2:1 ORFs, we constructed vectors carrying a mutant initiation codon of the green fluorescent protein (GFP) that was fused downstream of the full lnc-MRPL39-2:1 transcript or potential ORFs . GFP expression was observed in cells transfected with a wild-type GFP overexpression vector, but not in cells transfected with a mutant construct . Furthermore, western blot analysis confirmed that the lnc-MRPL39-2:1 ORFs were not successfully translated into proteins . Thus, we concluded that the lnc-MRPL39-2:1 has no protein-coding ability and is detected primarily in the cytoplasm of NPC cells.Analysis of different cell lines showed higher expression of lnc-MRPL39-2:1 expression in different cell lines compared to that in a non-cancerous human nasopharyngeal epithelial cell line (NP69) . Lnc-MRPL39-2:1 knockdown significantly inhibited NPC cell migration and invasion . Furthermore, the researchers conducted the MTS and EdU assays revealed that the NPC cell proliferation was significantly attenuated following lnc-MRPL39-2:1 knockdown . Colony-forming assays showed that lnc-MRPL39-2:1 knockdown decreased colony-formation in the NPC cells . Moreover, lnc-MRPL39-2:1 knockdown significantly increased NPC cell apoptosis . Overexpression of lnc-MRPL39-2:1 in transfected in NPC cells was confirmed by qPCR analysis . Lnc-MRPL39-2:1 overexpression markedly enhanced cell invasion, proliferation, and the clonogenic capacity of NPC cells . However, its overexpression decreased the number of apoptotic NPC cells and enhanced the radio-resistance of NPC cells . In addition, lnc-MRPL39-2:1 overexpression promoted cell invasion, proliferation, and the colony-formation, and decreased apoptosis of the human immortalized nasopharyngeal epithelial cell line NP69 in vitro . In conclusion, these results showed that lnc-MRPL39-2:1 increased the proliferative and metastatic capacity of NPC cells in intro.To validate the roles of lnc-MRPL39-2:1 in NPC cells, stable lnc-MRPL39-2:1-knockdown CNE2 and HONE1 cell lines were established . Lnc-MRPL39-2:1 was shown to bond directly to a 25-40 kD protein. Approximately 130 proteins were detected by MS analysis of the corresponding band, including the RNA binding protein (RBP) HuR, which is involved in the post-transcriptional regulation of cancer-related gene expression and interacts directly with lnc-MRPL39-2:1 . Immunohistochemical (IHC) analysis showed a higher level of HuR in NPC tissues than in para-tumor tissues. Moreover, patients with a high HuR expression had a shorter OS and PFS compared to those with low HuR expression . Next, lnc-MRPL39-2:1 was predicted to bond directly to the HuR protein by the RPISeq software. The direct interaction between lnc-MRPL39-2:1 and HuR was further verified by western blot of the lnc-MRPL39-2:1 pulldown cell lysate using an anti-HuR antibody and RNA immunoprecipitation (RIP) assay . Furthermore, to determine the HuR binding site within lnc-MRPL39-2:1, different deletion fragments were constructed based on their secondary structure as predicted by the RNA fold Web server. RNA pull-down and western blot assays indicated that a 1032-1659 nt region of lnc-MRPL39-2:1 was required for the interaction between lnc-MRPL39-2:1 and HuR . Finally, to determine the role of HuR in the tumor-promoting effects of lnc-MRPL39-2:1, CNE2 and HONE1 cells were transfected with lnc-MRPL39-2:1-overexpression vector and si-HuR. HuR knockdown partially abolished the lnc-MRPL39-2:1-induced proliferation and metastasis of NPC cells . Furthermore, the lnc-MRPL39-2:1-mediated inhibition of NPC cell apoptosis was rescued by si-HuR . Thus, we concluded that the lnc-MRPL39-2:1 promoted NPC cell growth and metastasis by directly binding to the HuR.To identify the proteins interacting with lnc-MRPL39-2:1 in NPC cells, we performed RNA pulldown assay with biotin-labeled lncRNAs (P < 0.05) in lnc-MRPL39-2:1 overexpressed CNE2 cells compared to the control group . KEGG analysis indicated that these differentially expressed genes were enriched in the JAK-STAT signaling pathway, Wnt signaling pathway, cell cycle, and the P53 and MAPK signaling pathway . These results revealed that lnc-MRPL39-2:1 may be a vital regulatory lncRNA involved in regulating NPC cell progression. Beta-catenin was one of the most upregulated factors. Previous studies confirmed that HuR can interact directly with the β-catenin mRNA to enhance both its half-life and expression ). Next, we performed RIP assays to examine the role of lnc-MRPL39-2:1 in the interactions between HuR and β-catenin mRNA. Lnc-MRPL39-2:1 was shown to interact directly with HuR and enhanced the interaction between the HuR and β-catenin mRNAs . Lnc-MRPL39-2:1 overexpression increased the levels of beta catenin. However, the lnc-MRPL39-2:1 expression was not decreased by β-catenin knockdown, indicating that β-catenin is a downstream effector of lnc-MRPL39-2:1 . Moreover, β-catenin silencing abrogated the ability of lnc-MRPL39-2:1 to promote NPC cell proliferation and metastasis in NPC cells . Furthermore, β-catenin knockdown increased NPC cell apoptosis . In conclusion, these results indicated that β-catenin is required in the mechanism by which lnc-MRPL39-2:1 promotes NPC proliferation and metastasis.Next, we performed RNA sequencing to determine downstream targets potentially involved in the mechanism by which lnc-MRPL39-2:1 promoted NPC cell growth and metastasis. Our analysis revealed 890 significantly up-regulated and 247 down-regulated mRNAs , we identified several miRNAs predicted to interact directly with lnc-MRPL39-2:1 (Table S3). Moreover, results from bioinformatic analysis predicted that miR-329 might bind to lnc-MRPL39-2:1 at two putative complementary sequences . RIP experiments showed that both lnc-MRPL39-2:1 and miR-329 were enriched in the AGO2 antibody-associated complex, indicating that miR-329 could directly interacts directly lnc-MRPL39-2:1 . Moreover, we found that miR-329 decreased the activity of the luciferase activity in cells transfected with the wild-type lnc-MRPL39-2:1 construct , and the MS2-RIP assay indicated that miR-329, but not the negative control miR-124, bound to the lnc-MRPL39-2:1 at the targeting site .Cytoplasmic lncRNAs function as ceRNAs by effectively binding to specific miRNAs and act as ceRNAs Fig. 6E). Moreover, miR-329 overexpression shortened the lnc-MRPL39-2:1 half-life in CNE2 and HONE1 cells . Furthermore, we demonstrated the involvement of miR-329 in the mechanism by which lnc-MRPL39-2:1 promoted NPC cell growth and metastasis by co-transfecting NPC cell lines with an lnc-MRPL39-2:1 plasmid with or without miR-329 mimics. MiR-329 overexpression inhibited the ability of lnc-MRPL39-2:1 to promote tumor growth and invasion . The negative correlation between the lnc-MRPL39-2:1 and miR-329 expression in NPC tissues was confirmed by qPCR . Thus, the carcinogenic effect of lnc-MRPL39-2:1 was shown to be partially mediated by the negative regulation of miR-329.Further experiments to determine the regulatory relationship between lnc-MRPL39-2:1 and miR-329 showed that miR-329 suppressed lnc-MRPL39-2:1 expression, whereas lnc-MRPL39-2:1 overexpression did not affect miR-329 expression, indicating that lnc-MRPL39-2:1 is targeted by miR-329 . We also examined the in vivo effects of lnc-MRPL39-2:1 overexpression on cell proliferation by carrying out IHC and hematoxylin and eosin (H&E) staining. Tumor cells invasion was observed in the epidermal appendages in the lnc-MRPL39-2:1 group but not in the control group. The relative ratio of Ki67-expressing cells was also higher in the lnc-MRPL39-2:1 group compared to that in the vector only group . Finally, in the lung metastasis model, we detected a higher total number of metastatic lesions in the lnc-MRPL39-2:1 overexpression group compared with that in the control group . Thus, our findings indicate that lnc-MRPL39-2:1 contributes to NPC tumorigenesis and metastasis in vivo.Having observed the pivotal role of lnc-MRPL39-2:1 in promoting NPC growth and invasion in vivo and in vitro. Additionally, lnc-MRPL39-2:1 expression could be suppressed by miR-329.To improve the poor prognosis of patients with NPC, it is necessary to decipher the molecular mechanisms involved in its recurrence and metastasis. Various genomic and epigenomic mechanisms are being investigated, with lncRNAs considered to play crucial roles in cancer proliferation and metastasis. In this study, we explored the roles of lncRNAs in NPC and the underlying mechanism. We identified a novel oncogenic lncRNA, lnc-MRPL39-2:1 that was upregulated in NPC tissues and associated with advanced tumor stage and poor prognosis of patients. Mechanistically, lnc-MRPL39-2:1 induced the growth and invasion of NPC by interacting with HuR to upregulate β-catenin expression, both To date, tens of thousands of lncRNAs have been identified and they were found to play crucial roles in different types of cancers. Moreover, several studies have demonstrated that dysregulated lncRNAs regulate NPC progression Nuclear lncRNAs and their functions have been studied quite extensively. However, cytoplasmic lncRNAs, which are not well understood, can also regulate protein localization and turnover and mRNA translation and stability. Some studies have shown that lncRNAs promoted the invasion and progression capacities of the NPC cells by regulating some critical targets, such as ITGB3, CDK4/6, or KAT2A acetyltransferase  et al. reported that HuR increased colorectal cancer growth and oxaliplatin resistance by regulating CDC6  et al. showed that lincRNA-UFC1 binds HuR leading to upregulation of β-catenin expression at both the mRNA and protein levelsHuR participates in the post-transcriptional regulation of several cancer-associated genes associated with cell proliferation and metastasis. For instance, HuR stabilized snail mRNA by binding to the downstream factor of the mRNA, thus promoting metastasis of pancreatic cancer cells Emerging data have indicated that lncRNAs function as the miRNA sponges. For instance,  in vivo and in vitro. These findings indicate that lnc-MRPL39-2:1 is essential in NPC tumorigenesis and metastasis and highlighted its potential as a prognostic marker and therapeutic target for NPC. Although, our findings provide insights into the value of lnc-MRPL39-2:1 as a promising therapeutic target against NPC, further studies are required to develop precise strategies targeting lnc-MRPL39-2:1 signaling and clarify the role of lnc-MRPL39-2:1 in other cancers.To conclude, the results of this study demonstrated that lnc-MRPL39-2:1 is upregulated in the NPC tissues and accurately predicts a poor survival rate of NPC patients in an accurate manner. Furthermore, it was shown that the lncRNA attenuates the growth and invasion of NPC by interacting with HuR, leading to upregulation of β-catenin expression bothFreshly frozen NPC and adjacent tissue samples were acquired from the Affiliated Cancer Hospital & Institute of Guangzhou Medical University. To analyze disease prognosis, we examined 121 NPC cases between January 2008 and December 2010 at the Affiliated Cancer Hospital & Institute of Guangzhou Medical University. All NPC patients were being treated using a standard treatment protocol. The patients had signed a written and informed consent form before being enrolled in the study. Thereafter, the researchers approached the Affiliated Cancer Hospital & Institute of Guangzhou Medical University and obtained approval for the study from its Institutional Ethical Review Board.Differentially expressed lncRNAs in NPC tissue were measured with the help of the Arraystar Human LncRNA Array V3.0 . Briefly, total mRNA purified from the NPC tumor and para-tumor tissues was quantified using the NanoDrop ND-1000 technique. Then, mRNA was amplified and labeled according to the kit manufacturer's protocol . Then, the researchers purified the labeled RNAs and determined their quality. Then, they hybridized it onto the Human LncRNA Array v3.0. After washing the slides, they were scanned by a DNA microarray scanner . Finally, the differentially expressed lncRNAs and mRNAs between the tumor and para-tumor tissues were validated according to fold change filtering. Further, the researchers carried out the KO and GO analyses to investigate the role played by the lncRNAs in cancer.2.Four NPC cell lines, i.e., CNE1, CNE2, SUNE1, and HONE1, along with the human immortalized nasopharyngeal epithelial cell line NP69 were obtained from the Shanghai Institute of Cell Biology . Every new batch of cell lines underwent mycoplasma testing and analysis of short tandem repeats. All the cell lines were incubated in the RPMI 1640 medium  that contained 10% Fetal Bovine Serum , penicillin, and streptomycin  at the optimal temperature of 37 °C and with 5% COThe researchers performed the RIP assay using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit . Briefly, the cells that were transfected with or without a vector were lysed with lysis buffer  Triton X-100, and 10% (v/v) glycerol) that also contained 1 mM proteinase and phosphatase inhibitor cocktail , and the RNase inhibitor . Then, the primary antibodies or IgG  were introduced into the cell lysates and this mixture was incubated overnight at 4 °C. Magnetic beads (Invitrogen) that were washed with the immunoprecipitation lysis buffer were added to the cell lysate mixture and incubated for 1 h at 4 °C. Finally, the beads were harvested and incubated at 70 °C for reversing the crosslinks. The sample was centrifuged and the RNAs in the supernatant were validated by qPCR. The results are presented as a fold change.Total RNA was isolated from the CNE2 cells using TRIzol (Invitrogen) and processed following the Trizell manufacturer's protocol. Subsequently, the researchers synthesized the cDNA from the extracted RNA using a cDNA Amplification kit . Then, they carried out the 5′ RACE and 3′ RACE reactions using a 5′ Full RACE Kit  and 3′ Full RACE Core Set with PrimeScript RTase Kit , respectively. Finally, the researchers separated the PCR products by electrophoresing the samples on a 1.5% agarose gel and then verified the products based on sequence analysis.The expression of lnc-MRPL39-2:1 was measured in paraffin-embedded samples using an ISH kit  based on the manufacturer's protocol. For this assay, the researchers cut the paraffin-embedded tissues into 4 μm thick sections. Then, these slides were deparaffinized and deproteinated. The specimens were incubated in the prehybridization solution at 42°C for 2 h. They were then incubated with a Digoxin-labeled oligonucleotide solution  complementary to lnc-MRPL39-2:1 at 37 °C overnight. Next, these slides were washed and stained with 3,3′-Diaminobenzidine (Sigma-Aldrich) and hematoxylin solutions. Finally, the slides were imaged, and the stained loci were counted under a microscope . The probe sequences for lnc-MRPL39-2:1 were as follows: AGAGGTGCCCAGTCCAAGTGAAGGGCAAGAGTCTACATAC; AACTATTACTCTAAAATGAAAGAATTCCTTTAAATTGAAT; GTCTAGCTATGCGTCCAGCACAGGCAGACTATGATGCCTG. Antisense: CAAACCGGAGACTAGCGAGCAT. in vitro with T7 RNA polymerase . Next, the samples were treated using RNase-free DNase I (Roche) and purified with a RNeasy Mini Kit . Biotinylated RNA was mixed with protein from CNE2 cell extracts and streptavidin agarose beads and incubated at room temperature for 1 h. Subsequently, the beads were boiled in sodium dodecyl sulfate (SDS) buffer and the pull-down proteins were analyzed by Western blotting. Finally, the SDS-polyacrylamide gel electrophoresis (PAGE) gel was silver-stained, and the important protein bands were extracted, cleaned, and analyzed by mass spectrometry (MS). Briefly, the gels were resuspended in digestion buffer with trypsin (Promega) and incubated for 20 h at 37°C. The peptides were then purified, dried, dissolved in 0.1% trifluoroacetic acid and validated using a Q Exactive mass spectrometer (Thermo). Trapping was performed at 10 μl/min for 4 min in 0.1% formic acid in water and the sample was loaded onto a reverse-phase column. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition. Finally, data analysis was performed using the Proteome Discoverer 1.4 software. The probes were as follows: TAATGTCCACTTGACCTCATCTATT; antisense: AATAGATGAGGTCAAGTGGACAT.RNA pull-down assays and deletion mapping were conducted as described previously -ΔΔCt technique. GAPDH and U6 were used as endogenous controls for quantifying the lncRNA and miRNA, respectively.The cells were transfected with siRNAs or plasmids with the help of the Lipofectamine 2000 reagent (Invitrogen) following the manufacturer's protocol. To check if the transfection was successful, the RNA expression was analyzed by qPCR. Briefly, we harvested total RNA from the cells or tissues using the TRIzol reagent (Invitrogen). Then, RNA was transcribed into complementary DNA (cDNA) with a cDNA Synthesis Kit (Promega). Expression analysis of RNA was carried out on an ABI 7500 PCR instrument using the SYBR Green PCR reagents  following the manufacturer's instructions. The researchers calculated the fold change by the 2The FISH Kit and lncRNA FISH Probe Mix  were used for FISH assays following the manufacturer's instructions . Briefly, cells seeded onto the slides in 24-well culture plates were fixed in 4% paraformaldehyde at room temperature for 30 mins. Cells were then cultured with a lncRNA probe and then counterstained with DAPI. Finally, cells were observed and images were captured on the laser scanning confocal microscope . In this experiment, the researchers used U6 and 18S RNA as the controls for the nucleus and cytoplasmic subcellular localization, respectively.3 cells/well. MTS solution was added to every well, and after incubation at room temperature for 3 h, the absorbance of every well was detected at 490 nm, and the relative cell viability was calculated.For the colony formation assay, NPC cells  were transfected with a vector containing the lncRNA or the control vector and seeded into the 6-well plates at the density of 1000 cells/well. The cells were allowed to grow for two weeks without disturbance. Finally, the colonies were fixed using methanol and then stained using crystal violet. The stained colonies were scanned by a high-resolution scanner, and the total number of colonies was quantified in each well. For the MTS assay, cells transfected with the lncRNA-expressing vector were introduced into the 96-well plates with 100 μL Dulbecco's modified eagle medium containing 10% FBS at the density of 5 × 10oC for 30 min. Subsequently, the cells were harvested and analyzed with the aid of flow cytometry.For the Click-iT® Plus EdU proliferation assay, cells were introduced into the 96-well plates and grown to 50-60% confluency. Then, cells were incubated with 10 µM EdU for 2 h. They were then reincubated with 100 µL 1× Apollo dyeing reaction solution at 4 4/mL into the upper chamber with/without Matrigel matrix precoating. RPMI 1640 medium supplemented with 10% FBS was added to the lower chamber. After fixing with methanol, the cells in the upper chamber were removed and the cells migrated to the lower chamber were stained using crystal violet. Photomicrographs of the stained cells were acquired with an inverted fluorescence microscope .NPC cells in the exponential growth phase were seeded into 24-cell culture plates and incubated in RPMI 1640 medium supplemented with 5% (v/v) FBS until a confluent cell monolayer was formed. A wound was incised in the central area of the culture plate using a 10-μL pipette tip, and the detached cells were removed. The researchers then added the RPMI 1640 medium with a low serum to the plates and images of the wound closure were captured at 0, 24, and 48 h using an inverted microscope . Finally, the wound width was analyzed with ImageJ software . Migration and invasion assays were performed using Transwell chambers . Transfected cells in serum-free RPMI 1640 medium were seeded at 5 × 10g for 12 min at 4 °C. Subsequently, the supernatant was harvested, and the total protein concentration was estimated using the BCA Protein Assay Kit . An aliquot (20 µg) of the total protein sample was separated by SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane . The PVDF membrane was blocked with 5% non-fat dried milk in Tris-buffered saline with Tween-20 (TBST) and incubated in the presence of primary antibodies  overnight at 4°C. The membrane was then washed with TBST buffer and incubated with the corresponding HRP-conjugated secondary antibody for 1 h at room temperature. Finally, immunoreactive signals were visualized using the ECL reagent (Merck Millipore).Cells were suspended in the lysis buffer that contained a protease inhibitor cocktail (Sigma-Aldrich) and centrifuged at 15,000 × For lncRNA-MRPL39-2:1 and miR-329 luciferase assays, lncRNA-MRPL39-2:1 sequences containing wild-type (WT) or mutant (Mut) miRNA binding sites were synthesized and inserted into the pmirGLO luciferase vector . The constructs were then co-transfected with miR-329 mimics into HEK293T cells using Lipofectamine3000 (Thermo Fisher). After 48 h, relative luciferase activity was measured using the dual-luciferase reporter gene system (Promega) according to the manufacturer's protocol. Relative luciferase activity was normalized to the Renilla luciferase internal control.https://www.ncbi.nlm.nih.gov/geo/). The expression of lncRNA-MRPL39-2:1 in tumor and non-tumor ovarian samples from the databases were analyzed using the R package.NPC‐related gene expression data set gene set enrichment (GSE) 53819 included 18 NPC tissue specimens and 18 non-cancerous tissue specimens. The array data for GSE12452 contains 41 samples, including 31 NPC and 10 normal nasopharyngeal tissues. Both datasets were retrieved from the Gene Expression Omnibus  was utilized to evaluate the statistical significance of the enrichment of differentially expressed mRNAs and lncRNA target genes in KEGG pathways (Kyoto Encyclopedia of Genes and Genomes). Two-sided or Bonferroni-corrected Fisher's exact tests were used to calculate the P-values of KEGG pathways or GO terms, respectively. A threshold of P < 0.05 and FDR ≤ 0.25 was applied to select significant items.The functional roles of the differently expressed mRNAs and lncRNA target genes were investigated by GO enrichment analysis using the GOseq R package. DAVID software (https://lncipedia.org/), DIANA tool (http://diana.imis.athena-innovation.gr) and TargetScan (http://www.targetscan.org) were used to screen potential miRNAs that may bind with the lnc-MRPL39-2:1 transcript.The PhyloCSF developed at the University of California Santa Cruz (UCSC) was utilized to analyze the coding potential of lnc-MRPL39-2:1 transcripts. Three other online software packages, LNCipedia, RegRNA 2.0 and the Coding Potential Assessment Tool (CPAT), were also used for potential noncoding analysis. The online LNCipedia (The tissue samples from humans or mice were fixed in 4% (v/v) paraformaldehyde solution for 48 h. Then, the 4 μm-thick paraffin-embedded tissue sections were deparaffinized using xylene and rehydrated using a graded ethanol series. The sections were incubated in the blocking solution for 10 mins and then incubated again in the presence of corresponding primary antibodies at 4 °C overnight. Next, these sections were incubated using secondary antibodies for 1 h, counterstained with hematoxylin (Sigma-Aldrich), and imaged by a light microscope . The percentage of the positive cells and staining intensity were analyzed by two independent observers. in vivo, the researchers purchased six-week-old female BALB/c(nu/nu) nude mice from the Guangzhou Medical University. The researchers investigated the lnc-MRPL39-2:1-induced tumor growth in vivo by means of subcutaneously injecting lentiviral-infected NPC cells (5×106/100 µL) into the right upper flank of the mouse, and measured the tumor volume every 3 days for 30 days. To validate the metastatic ability promoted by the lnc-MRPL39-2:1, cells infected with lentivirus carrying only vector or lnc-MRPL39-2:1  were injected into the tail veins of the mice, and the tumors formed in the lung were monitored using a bioluminescence imaging system. Finally, six weeks after injecting the cells, the mice were euthanized, and the number of tumors in the lung was observed and stained with H&E.The animal experiments were approved by the Medical Ethics committee at Guangzhou Medical University . To validate the tumorigenesis and metastasis of lnc-MRPL39-2:1in vitro experiments were expressed as the mean value ± SD based on three or more independent experiments. The researchers employed the Kaplan-Meier technique and Cox proportional hazard regression model for the purpose of analyzing the OS. They further used the chi-square test and Fisher's exact test for analyzing the relationship between the lncRNAs and other clinicopathological parameters, including age, clinical stage, and tumor size. A P-value<0.05 was considered to be statistically significant.Statistical analyses were carried out using the SPSS ver. 23.0 software . All data acquired from the Supplementary figures and tables.Click here for additional data file.This work was supported by the National Natural Science Foundation of China , the Guangzhou Key Medical Discipline Construction Project Fund, and the Key Clinical Technology of Guangzhou (2019D17).


Neurosarcoidosis tends to prolong the duration of treatment and may result in a decline in physical function requiring rehabilitation. Because of a rare disease, the adjustment of oral steroid dosage, which is the cornerstone of treatment, is highly dependent on professional experience in general. Therefore, the number of hospitals that can perform dosage adjustment is very limited, and it is difficult to provide concurrent intense rehabilitation at the same hospital over a long period of time, and there are no reports that mention this.A 49-year-old man, who presented with impaired consciousness, dysphagia and right hemiplegia, was diagnosed with neurosarcoidosis based on a previous diagnosis of sarcoidosis, laboratory test results, and clinical symptoms. High-dose oral steroid therapy was initiated and he was transferred to our rehabilitation hospital for progressive disuse approximately 2 months after the disease onset.This case was diagnosed as “probable” neurosarcoidosis.The steroid dose was not reduced during rehabilitation treatment in our hospital considering the risk of relapse of the primary disease due to steroid reduction. His training regimen focused on minimum activities of daily living was performed, and its effectiveness was determined during approximately 60 days after the initiation of rehabilitation.Two months after admission, he was independently eating, transferring, and toileting under supervision. He was discharged home 3 months after admission.Intensive rehabilitation can be one of the effective comprehensive treatment strategy for patients with neurosarcoidosis. On the other hand, since there is no consensus treatment method, the duration of rehabilitation and goal setting should be adjusted based on an understanding of the characteristics of the disease and the overall treatment plan. Sarcoidosis affecting the central nervous system is often treated with potent immunosuppressive therapy. However, because evidence-based treatment guidelines for treatment do not exist, careful handling of each case is necessary. Treatment tends to be prolonged, and patients may require rehabilitation to address the decline in physical function and other disease complications. On the other hand, because of the heavy reliance on the experience of specialists, only a limited number of hospitals are able to adjust the dosage of oral steroids, the cornerstone of treatment. In other words, patients with neurosarcoidosis who require treatment long enough to require rehabilitation must choose between continued dosage adjustment and intense rehabilitation therapy. Unfortunately, few reports mention about this problem.Sarcoidosis is an immune-mediated disease of unknown cause characterized by granulomatous inflammation of various organs, including the lungs in 90% of cases, skin (15%), eyes (10–30%), liver (20–30%), and lymph nodes (10–20%).This case report discusses the potential of rehabilitation therapy as one of the comprehensive treatments for neurosarcoidosis.A 49 year-old man developed anterior chest discomfort and respiratory distress at the age of 28 years, and was diagnosed with sarcoidosis via a lymph node biopsy in the hilar region of the lung revealed a noncaseating granuloma. Then he was prescribed a low dose of steroids. Diabetes mellitus was noted after the initiation of treatment for sarcoidosis. Moreover, at 39 years old, he suffered a cerebral infarction, resulting in left hemiplegia; however, he was able to live independently with the use of a short leg brace.4/µL, angiotensin converting enzyme 8.1 U/L , IgG 756 mg/dL , soluble interleukin-2 receptor 444U/mL . A lumbar puncture revealed an elevated initial pressure of 290 mm H2O . A cerebrospinal fluid (CSF) examination revealed protein 225 mg/dL , glucose 57 mg/dL , and cell count 29/μL , of which 94% were mononuclear cells. Angiotensin converting enzyme in the CSF was 1.2 U/L, and soluble interleukin-2 receptor in the CSF was 386 U/ml. Oligoclonal bands were negative, and the IgG index was 0.51 . CSF cultures of bacteria and Mycobacterium tuberculosis were negative, as were polymerase chain reaction tests for human herpesvirus and varicella-zoster virus, indicating negative results for meningitis, encephalitis, and other infections. Chest computed tomography revealed a swollen mediastinal lymph node , including standing and sitting Table , with a There is no evidence-based treatment for neurosarcoidosis, and the following 3 policies in current patient’s rehabilitation treatment were shared with the patient. First, steroid medication should not be adjusted during intensive rehabilitation considering the risk of relapse of the primary disease due to steroid reduction. Second, to facilitate discharge, the goal is to focus on achieving the minimum necessary activities at home, such as transferring, toileting, and eating. Finally, 1 to 2 months after admission, it should be determined whether a patient can achieve the target physical function. Moreover, if a patient cannot achieve the goals, he should be transferred to a general hospital with a neurology department, where pharmacotherapy can be prioritized.Since he was taking high-dose steroids, blood glucose control was strictly maintained, masks were worn at all times as part of infection control measures, and training was limited to the same ward. At the beginning of the rehabilitation therapy, training focused on strengthening trunk and lower limb muscles and stabilizing the sitting posture, and encouraged the patient to move away from the bed firstly.On the other hand, intermittent muscle tonicity was observed at admission in the patient’s right upper and lower limbs. This muscle tonicity was challenging to control voluntarily and interfered with basic movements, such as standing. Since the muscle tonicity extended to the face, he was considered to have epileptic seizures related to neurosarcoidosis, and carbamazepine was started at 200 mg/day on the 60th day of illness. After that episode, his involuntary movements decreased dramatically, and he could perform the daily rehabilitation exercises well. Around the 80th sick day, he stabilized his sitting posture, although he required assistance while standing up. Considering that involuntary movements of the right lower extremity might have affected his standing and walking activities, a short leg orthosis was made for the right lower limb on the 94th sick day. By the 100th sick day (2 months after admission), he could sit independently and transfer and use the toilet under supervision. It was hoped that he could gain physical functions as initially intended (Table  but the condition does not have definitive laboratory findings. In addition, biopsies of nerve tissue are generally tricky, making the diagnosis very difficult.,6 Therefore, it is important to diagnose patients with systemic sarcoidosis that present with neurological symptoms comprehensively, including laboratory findings. Based on the neurological and CSF findings and the previous diagnosis of sarcoidosis, this case was determined as “probable” neurosarcoidosis. The improvement in neurological symptoms after treatment with steroids further supports the diagnosis of neurosarcoidosis. In addition to multiple cranial neuropathies, the current patient had impaired consciousness, right hemiplegia, and convulsive seizures, suggesting that the peripheral nervous system and the central nervous system were affected. Furthermore, based on the variety of cortical symptoms, the lesion was considered a diffuse disseminated intraparenchymal granuloma lesion.Neurosarcoidosis presents with various symptoms depending on the site of involvement,,8 case reports are the primary source of information on its clinical spectrum. Furthermore, treatment strategies are limited to expert opinion. Therefore, there have been few reports of rehabilitation treatment as one of the comprehensive treatment measures in parallel with drug therapy, as in the present case.Because neurosarcoidosis is a relatively rare disease,,11 Since the method of dose reduction depends on the experience of the treating physician, steroid dose reduction should have been carefully performed in a specialized department. In previous reports, there were cases in which the steroid dosage was not changed during rehabilitation intervention.,12 Steroid dose reduction should be avoided in parallel with rehabilitation therapy because the symptoms of this rare condition cannot be adequately managed in this rehabilitation hospital setting. Second, many cases of diffuse disseminated intraparenchymal granuloma lesions require long-term oral steroid therapy, as in this case. Such long-term, high-capacity steroid therapy is associated with an increased risk of developing steroid-related side effects and severity.,10 In this case, because the patient continued to receive prednisone at the starting dose, he had to be adjusted to the lowest effective dose after rehabilitation therapy. Therefore, the purpose of limiting the duration of rehabilitation was to prevent steroid-associated side effects and to facilitate the prompt dose adjustment of the oral medication, the mainstay of treatment, as part of a comprehensive strategy.In light of the above, the following points should be noted when considering the overall treatment plan in this case. First, neurosarcoidosis treatment is an aggressive immunosuppressive therapy centered on steroids, but the clinical symptoms may worsen if steroids are reduced. On the other hand, some argue that the pathophysiology of epileptic seizures and neurosarcoidosis do not necessarily coincide, and the appearance of epileptic seizures does not necessarily reflect worsening pathophysiology. Therefore, anticonvulsants should be used aggressively when epileptic seizures inhibit rehabilitation.This patient responded well to steroids, and symptoms such as impaired consciousness and hemiplegia improved promptly. However, his disuse progressed because of the prolonged treatment period. Therefore, the main focus was on rehabilitation treatment for the deterioration of physical function due to inactivity while managing diabetes, taking infection control measures, and providing countermeasures against epileptic seizures. In particular, in systemic management, the impact of anticonvulsants on the pathophysiology of neurosarcoidosis is still poorly understood.,12,14 However, all of them were interventions in the acute phase, and the duration of intervention was relatively short. The following factors were cited as contributing to a favorable outcome: early high-dose steroid administration, young age, good health before onset, and early implementation of intensive rehabilitation. This case differs from previous reports in that the patient started intensive rehabilitation more than 50 days after onset, and the treatment strategy differs from previous cases in that the maximum duration of rehabilitation was determined at the start as part of a comprehensive treatment strategy, and drug therapy for symptoms associated with neurosarcoidosis was provided by a single department of rehabilitation medicine. In other words, even if it is not possible to start intensive rehabilitation at an early stage, as long as the underlying disease is controlled by drug treatment, effective rehabilitation can be provided, leading to recovery of physical function. This approach indicates that intensive rehabilitation may be an option for comprehensive treatment for patients with neurosarcoidosis who require long-term treatment.A few case reports describe improved physical function following rehabilitation treatment in patients with neurosarcoidosis.Neurosarcoidosis is a rare disease with no consensus treatment plan or long treatment period. Intensive rehabilitation therapy, combined with an accurate plan based on a thorough knowledge of the treatment strategy and clinical manifestations of neurosarcoidosis, may reduce complications from steroids and improve the patient’s ADL and subsequently maintain quality of life.The authors would like to express our deep appreciation to Kayo Takeshima for her great effort in preparing this report.Conceptualization: Shinichi Takeshima.Data curation: Shinichi Takeshima, Toshiki Furuya, Mariko Yamamoto, Marie Noma.Project administration: Nobuyuki Kawate.Supervision: Nobuyuki Kawate.


Allergic rhinitis (AR) is a common condition that can be treated with a number of different therapies. Treatments such as intranasal antihistamines (INAs) and intranasal steroids (INSs) are widely used by AR patients. For some allergy sufferers, a combination of therapies, specifically an INA and an INS, is required to address their symptoms. A new treatment, the formulation of azelastine hydrochloride and fluticasone propionate used as a single spray (MP-AzeFlu), has become available for AR patients who need both types of treatment. In this regard, the comparison with the alternative concomitant use of INAs and INSs is of interest. The current study examines the health care resource utilization and costs for each cohort.To examine the resource utilization and costs associated with AR for patients treated with MP-AzeFlu or concurrent therapy with single-ingredient INA and INS sprays (free-combination therapy).A retrospective administrative claims study for commercially insured patients from a large U.S. health plan was performed. Patients with an AR diagnosis and a prescription claim for MP-AzeFlu or free-combination therapy between September 1, 2012, and September 30, 2013, were identified. Patients were aged at least 12 years at index date  and were required to have 12 months pre-index and 6 months post-index of continuous enrollment. Health care resource utilization and costs were assessed for the post-index period. The cohorts were adjusted on baseline demographic and clinical characteristics using inverse propensity treatment weights. Other covariates, prescriber specialty, product switching during the post-index period, and pre-index total costs were included in the regression models measuring outcomes. One clinical characteristic of interest was the presence of asthma as comorbidity. A subset analysis of AR patients with asthma was also performed.P < 0.001) with no significant difference in outpatient services and specialist visits . Six-month AR-related pharmacy and total costs were significantly lower (P < 0.001 and P = 0.001) for the MP-AzeFlu cohort  than the free-combination cohort . There was no statistically significant difference in AR-related medical costs between the 2 cohorts (P = 0.454). For the subcohort of AR patients with asthma, the MP-AzeFlu cohort had lower 6-month asthma resource utilization and costs than the free-combination cohort.All-cause-related pharmacy fills as well as pharmacy, medical, and total costs were significantly reduced by using MP-AzeFlu (N = 810) instead of the free combination of drugs (N = 726). For AR-related health care resource utilization, the MP-AzeFlu cohort had significantly fewer pharmacy fills than the free-combination cohort (1.01 and 1.17, respectively; These findings suggest that, for AR patients needing INAs and INSs, the single-spray formulation MP-AzeFlu had better economic outcomes than for patients who rely on the free combination of these agents. MP-AzeFlu also appears to keep asthma-related utilization and costs down for those AR patients who also suffer from asthma. Potential explanations for these findings are explored. What is already known about this subjectAllergic rhinitis (AR) is one of the most common chronic conditions in the United States, with close to 38% of patients also suffering from asthma.Successful management of AR can be a challenge and can lead to dissatisfaction with the more common pharmacotherapies .MP-AzeFlu is a safe and effective treatment of AR in patients who require dual therapy with an INS and an INA.What this study addsThis study compared MP-AzeFlu with free-combination therapy and examined the use of MP-AzeFlu and its effect on health care resource utilization and costs for patients with AR and those with concurrent asthma.AR patients prescribed MP-AzeFlu had similar or less utilization than patients taking a combination of INA and INS drugs (free combination); pharmacy costs and total costs were lower for patients on MP-AzeFlu, while AR-related medical costs were similar to those on the free combination of therapies.For AR patients also diagnosed with asthma, those prescribed MP-AzeFlu had similar use of emergency department services, outpatient services, and specialist visits, but significantly fewer pharmacy fills than those prescribed a free combination of INA and INS products.4 The prevalence of AR has been on the rise for many years, and it is currently the sixth most common chronic disease in the United States, leading to $18 billion of medical costs annually.5Allergic rhinitis (AR), characterized by sneezing, nasal congestion, nasal itching, and rhinorrhea, is one of the most common chronic conditions in the United States, affecting approximately 22% of adults and up to 40% of children.9 Asthma is a common comorbid condition in patients with AR.10 In the United States, up to 38% of AR patients suffer from asthma, and as many as 90% of patients with asthma suffer from concurrent AR.2 It is unclear whether AR is a trigger or component cause for asthma, but AR is a source of diminished quality of life for individuals with asthma. Additionally, in individuals with both conditions, AR is associated with poorly controlled asthma, including more frequent asthma exacerbations and asthma-related emergency department (ED) visits, while treatment of AR is associated with fewer asthma-related hospital admissions and ED visits.14Not surprisingly, AR has a significant detrimental impact on the quality of life of afflicted individuals as well as leading to substantially increased health care costs.15Most cases of AR respond to pharmacotherapy, including oral antihistamines, decongestants, intranasal steroid (INS) sprays, intranasal antihistamine (INA) sprays, and immuno-therapy. INSs and INAs are the primary medical therapies used and may alternatively be used as first-line monotherapy agents for patients with intermittent or mild persistent forms of AR.16 Escalation of medical management to include combination therapies is recommended for patients with more difficult to control disease, such as those with moderate-to-severe, persistent AR.15Despite the wide variety of therapies that exist, successful management of AR remains a challenge. Inadequate symptom control can often lead to dissatisfaction in patients taking prescription AR treatments, resulting in poor medication adherence or increasing reliance on over-the-counter medication.15 Previous research has demonstrated that this new MP-AzeFlu formulation has an excellent safety profile and has been shown to be more effective in controlling AR symptoms than other first-line therapies.17, 18 While previous studies have examined the clinical effectiveness of MP-AzeFlu compared with other therapies, there is a paucity of research on the impact of this AR treatment regimen on health care resource utilization and costs.Until recently, patients needing a stronger pharmaceutical treatment than INSs had limited options. A new treatment, the formulation of azelastine hydrochloride and fluticasone propionate used as a single spray (MP-AzeFlu), has become available for AR patients who need treatment with an INS and an INA.20 We focus specifically on health care utilization and costs related to AR and asthma in AR patients treated with either MP-AzeFlu or the free combination of INS and INA.The purpose of this study was to determine health care utilization and costs of patients with AR using MP-AzeFlu in comparison with those patients using the potential alternative free combination of INS spray and INA spray. In order to investigate this relationship, we performed a retrospective claims-based study of commercially insured individuals. Previous studies have likewise used insurance claims data to compare resource utilization and costs for medical treatments of AR.International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) associated with medical encounters and financial information. Pharmacy claims included fill dates for prescriptions, national drug codes, and drug cost data. The current study was submitted and approved by Schulman Associates Institutional Review Board before study initiation.All data for the current study were from the Humana administrative claims database between September 1, 2011, and March 31, 2014, for commercially insured patients. The database contained patient enrollment and medical and pharmacy claims data. Enrollment data included patient demographics such as age, sex, and geographic region. Medical claims data included diagnosis codes based on the Two cohorts of commercially insured patients were identified based on the type of intranasal treatment prescribed— MP-AzeFlu or free-combination therapy (INS and INA). Patients in both cohorts had to have a prescription claim for either MP-AzeFlu or free-combination therapy between September 1, 2012, (corresponding to the month of launch of MP-AzeFlu) and September 30, 2013, with 1 claim for a diagnosis of AR (ICD-9-CM: 477.xx) or chronic rhinitis (ICD-9-CM: 472.0) between September 1, 2011, and March 31, 2014. The first prescription claim identified for patients was designated as their index date. The index date for the MP-AzeFlu cohort consisted of the first prescription claim for that treatment, while the free-combination therapy cohort index date was the first prescription claim for an INA and an INS that occurred within 7 days of each other or showed an overlap of prescriptions for 90 days. All patients included in the current study had to be newly initiating on either of the therapies. This meant there could be no claim for an intranasal treatment prior to the index date for any participant to be included. To ensure study patients had no prior intranasal therapy claims, inclusion criteria included a 12-month period of health care coverage with no prescriptions and pharmacy fills for intranasal therapy.Further inclusion criteria consisted of patients being between the ages of 12 and 89 years and having at least 12 months pre- and 6 months post-index continuous enrollment. Patients were excluded from either cohort if they had 1 claim for a diagnosis at any point during the identification period  for endstage renal disease (ICD-9-CM: 585.6), cancer (ICD-9-CM: 140.xx-172.xx and 174.xx-208.xx), pregnancy , or organ transplant (ICD-9-CM: V42.xx); were older than 89 years; or had any claims for an INS or an INA within 1 year of the index date. Patients aged younger than 19 years and residing in Nebraska, Delaware, or Alabama were also excluded due to laws in those states prohibiting research using patients younger than 19 years. Demographic and clinical characteristics were examined for the 12-month baseline period for patients in each cohort. Demographic characteristics included age, sex, and geographic locale. Geographic locale was further broken out by states with the largest plan populations  and then by regions . The season for each patient’s index date was also tracked as winter ; spring ; summer ; and fall .21 Other pre-index conditions that were flagged and tracked included asthma (ICD-9-CM: 493.xx), acute sinusitis (ICD-9-CM: 461.xx), chronic sinusitis (ICD-9-CM: 473.xx), sleep apnea (ICD-9-CM: 327.23 and 780.57), nasal polyps (ICD-9-CM: 471.xx), allergic rhinoconjunctivitis (ICD-9-CM: 372.14), and nonallergic conjunctivitis (ICD-9-CM: 372.05).Clinical characteristics included a Deyo-Charlson Comorbidity Index (DCCI) score that was calculated for each patient.All-cause and AR-related health care resource utilization were calculated using all post-index medical and pharmacy claims. The index event was excluded from the post-index period. For the AR- and asthma-related utilization, only those medical and pharmacy claims associated with a diagnosis of AR or asthma were used. A diagnosis for AR or asthma could appear in any of the 9 diagnoses positions within the claims data. However, more than 90% of the claims for AR for the cohort patients were found in the primary position. Health care utilization was calculated for all 3 breakdowns  for ED services, outpatient services, specialist visits, and pharmacy fills. ED services were assessed by counting the distinct service dates at an ED as defined by the place of treatment code found on the medical claim. Outpatient services included the count of distinct service dates and distinct providers for claims where the place of treatment is listed as outpatient. Specialist visits were a subset of outpatient visits where the provider is a specialist as specified by the physician specialty code on the claim. Pharmacy fills consisted of the count for distinct service dates (fill date) and national drug code. Pharmacy fills were normalized so that each fill could be equated to a 30-day supply. Furthermore, the INA and INS fill pairings for the free-combination cohort were counted as 1 fill for the index fill and all subsequent pharmacy fills. Both prescriptions were key for patients to be included in the cohort. By counting the combination of prescriptions as 1 fill, there would be no artificial representation by including each product as 1 fill.For AR-related utilization, medical utilization was defined as any claim containing a diagnosis code for AR ; chronic rhinitis (ICD-9-CM: 472.0); acute sinusitis (ICD-9-CM: 461.xx); or chronic sinusitis (ICD-9-CM: 473.xx); or if the claim line procedure code was associated with allergy testing  or allergy immunotherapy (CPT-4: 95115-95199). Pharmacy utilization was defined as any pharmacy claim for a product used to treat allergies .For asthma-related utilization, medical utilization was defined as any claim containing a diagnosis code for asthma (ICD-9-CM: 493.xx). Pharmacy utilization was defined as any claim for a product used to treat asthma (see Appendix for complete list).Similar to calculations performed for health care utilization, all-cause, AR-related, and asthma-related health care costs were calculated. Costs were examined for all medical, pharmacy, and total  costs. The allowable amount, defined as the amount remitted to the providers, found in the claims data was used in the analysis of health care costs for the current study. As in health care resource utilization, the index fill cost was excluded from the post-index period calculations with all postindex costs measured during the 6-month period following the index date for patients. All health care costs were adjusted to 2013 dollars, as it was the last complete calendar year for the study observation. The medical consumer price index is used for the adjustment.All analyses were conducted using SAS software package 9.3 . For the demographic and clinical characteristics, means and standard deviations were calculated for continuous variables. For any categorical variables, counts and percentages were calculated. For the demographic and clinical characteristics, comparisons between the cohorts were conducted using t-tests for all continuous variable data and chi-square tests for all categorical data.22 IPTW was used to balance the differential characteristics of patient cohorts. Age, sex, DCCI and other comorbid conditions, geographic region, and season were the characteristics that contributed to the weights used for the IPTW adjustment. Unadjusted (scores prior to matching) and adjusted (scores following matching) results are presented for demographic and clinical baseline characteristics. All health care resource utilization and cost outcomes presented are adjusted.To minimize the risk of selection bias for the 2 cohorts before computing any outcome measures, inverse propensity treatment score weighting (IPTW) was employed.Generalized linear models using a log-link function and gamma distribution were generated to assess health care costs, and linear regression models were generated to assess measures of health care utilization as our dependent variables and AR treatment (MP-AzeFlu vs. the free-combination therapy) as the independent variable. The demographic and clinical characteristics identified for patients in each cohort were included in the models as covariates. Least squares means were computed for both utilization and costs. Along with the IPTW adjustment, the least squares means for utilization and costs were also adjusted for prescriber type , switching status (whether the patient switched treatments during the post-index period), and pre-index total costs . Significance for all analyses was set at a 0.05 alpha level a priori.P = 0.255; outpatient services, P = 0.624). Patients in both cohorts had approximately 5 specialist visits during the 6 months following their AR diagnosis and index therapy prescription. However, patients in the free-combination cohort had significantly more pharmacy fills  following their index prescription than those using MP-AzeFlu (11.26).Health care utilization was examined using all medical and pharmacy claims  as well as for AR-related claims during the 6 months following patients’ index prescription. P = 0.139; specialist visits, P = 0.117). Neither cohort had claims for ED visits during the 6 months after filling the index therapy. Just as with the all-cause analysis, patients in the free-combination cohort had significantly more AR-related pharmacy fills post-index (1.17) than the MP-AzeFlu cohort .With respect to AR-related utilization, patients in the MP-AzeFlu cohort had slightly more outpatient services (2.13) and specialist visits (1.73) related to AR than the free-combination cohort , but this was not statistically significant . Total medical costs and total costs were significantly lower  for the MP-AzeFlu patients  than for the free-combination patients .Health care costs were likewise classified as all-cause or AR-related. These costs included all pertinent medical, pharmacy, and total  costs. P < 0.001) for the MP-AzeFlu cohort ($128) compared with the free-combination cohort ($268). If the cost of the nasal spray fills on the index dates is included, the mean AR-related pharmacy costs increase about $150—from $128 to $279 for the MP-AzeFlu cohort and from $268 to $418 for the free-combination cohort, and this cost differential between the 2 cohorts remains statistically significant (P = 0.048). AR-related medical costs were similar for the MP-AzeFlu patients ($206) and the patients in the free-combination cohort ($190) 6 months following their index prescription fill, and this was not statistically significant (P = 0.454). Total costs were also lower for the MP-AzeFlu cohort patients ($334) than for the free-combination cohort . This difference in total costs remains quantitatively similar and statistically significant even if the cost of the nasal spray fills on the index date is included.AR-related pharmacy costs were significantly lower . ED visits were similar across both cohorts. Free-combination patients also had significantly more prescription fills (3.35) than the MP-AzeFlu cohort patients (1.50) following the index day (P < 0.001).Patients in both cohorts with a concurrent diagnosis of asthma were examined separately for utilization and costs. Of the originally identified cohorts, 109 patients on MP-AzeFlu and 113 patients on the free-combination therapy also had a diagnosis of asthma. For these patients, asthma-related claims  were assessed and are reported in P = 0.008) for the free-combination cohort patients ($928) than for the MP-AzeFlu patients ($192). Total medical costs were low but similar for the MP-AzeFlu cohort ($351) and free-combination cohort . Total costs were significantly higher for the free-combination cohort  than for the MP-AzeFlu cohort .Total pharmacy costs were significantly higher  In order for any significant difference to occur between these 2 cohorts, there needed to be a large difference in the count of utilization services. While the free-combination cohort had higher means for ED visits and outpatient services, this did not equate to significantly higher utilization. However, the higher count of services would be associated with higher costs, since 1 ED visit can generate higher costs.Current research shows that there is an incremental increase in the utilization and costs for individuals diagnosed with AR.We additionally found that AR-related pharmacy costs were lower in patients on MP-AzeFlu than in patients on the free combination of INA and INS. Previous arguments made by third-party payers against the use of MP-AzeFlu were based on the immediate and direct cost associated with MP-AzeFlu relative to the free combination of INA and INS. However, this approach does not take long-term costs into consideration. We found that patients on the free combination of INA and INS had more prescription fills for their nasal sprays over our study period. Additionally, over time, AR-related pharmacy costs are only partially composed of either the cost of MP-AzeFlu or the free combination of an INS and an INA. AR-related pharmacy costs consisting of other medications such as decongestants or nasal treatments such as saline irrigations must also be considered.In our study, AR patients on the free combination of INA and INS may have used more expensive ancillary AR-related therapeutics to supplement their INA and INS, which led to higher overall AR-related pharmacy costs in comparison with patients using MP-AzeFlu. The decision was made to exclude the index fill cost from all analyses, as this helped to ensure a clearer examination of all post-index outcomes. While there are arguments to include the index fill, the only impact of including this cost was found for pharmacy utilization, where the fill count increased by 1 for both groups. The health care costs increased for both groups, but the cost differential between the groups was identical and the statistical difference was the same.26 One explanation for the finding that the MP-AzeFlu cohort had lower asthma-related pharmacy costs could be better control of AR by MP-AzeFlu, leading to better asthma control, although this remains speculation at this point and other explanations may exist.Interestingly, we also found lower asthma-related health care utilization and pharmacy costs in AR patients with asthma on MP-AzeFlu compared with those on the INA and INS free combination. The “unified airway hypothesis” suggests that upper and lower airway inflammation may be linked and that the exacerbation of one may in turn lead to exacerbation of the other.29 When examining utilization and costs, only 6 months of data were available for use, which may not be comparable to other studies on AR treatments that used 12 months of post-index data.This study has several limitations that should be considered when interpreting the current findings. These findings are only applicable to the members identified in the study population. Additionally, the analyses were only completed for those patients who had newly initiated to MP-AzeFlu or a free combination of products. The study did not examine patients in the Medicaid population, who have previously been shown to have different utilization patterns and severity of disease than patients with private insurance  or Medicare coverage.While there is a case to be made for using prevalent AR patients, by focusing on new initiators, confounders to subsequent utilization associated with the effects of previous nasal spray use were removed. The analyses performed here were also completed for all members, not just new initiators, and results were similar to those presented here.The severity of AR-related symptoms or asthma, which is not available in claims databases, could also not be accounted for in this study. The relative similarity in unadjusted frequency of atopic comorbidities such as acute and chronic sinusitis and nasal polyps between the MP-AzeFlu and free-combination cohorts suggests that on the whole these 2 cohorts were similar. However, the definitive lack of AR and asthma severity nonetheless potentially confounds our results. Also, as this is a retrospective claims-based study, based on the patterns of a select sample of patients, no causal inferences can be ascertained from the results. Because these results are limited to a specific cohort of commercially insured patients at a large health plan, they cannot be generalized to the larger, external population.Adherence was not an outcome of interest for this study, partly because patients’ utilization may vary as a function of the severity of the symptoms they experience, and the severity of AR is not available on claims data.Finally, pharmacy costs included for analyses were limited to the medications that require a prescription and could be found in the pharmacy claims, leaving over-the-counter medication treatments out.In a comprehensive claims database of commercially insured patients with AR requiring dual therapy with an INA and an INS, we found the cohort taking MP-AzeFlu had lower overall, AR-related, and asthma-related health care utilization and costs compared with a matched cohort taking the free combination of an INA and an INS. MP-AzeFlu may offer advantages with respect to medical costs, in particular, AR- and asthma-related pharmacy costs, over the free combination of INA and INS. At the very least, MP-AzeFlu does not appear to be associated with higher costs. This information should be considered when treating AR patients who require dual INS and INA therapy, in particular, those with asthma.


The objective of this study was to design and validate a nomogram of intranasal corticosteroid (INCS) insensitivity for adult patients with allergic rhinitis (AR).  Training and validation datasets comprised randomly divided groups of AR patients diagnosed between 2019 and 2022, with a 7 : 3 ratio. These patients were categorized according to their INCS insensitivity status, and LASSO and multivariate logistic regression analyses were conducted to identify associated risk factors. These factors were incorporated into a nomogram for predicting INCS insensitivity. The performance of the nomogram was assessed using receiver operating characteristic (ROC) curves, calibration curves, and discrimination techniques.  In this study, 313 patients were included, of which 120 (38.3%) showed INCS insensitivity. The type of AR, comorbidities, family history of AR, and duration of AR were identified as predictors and incorporated into the nomogram using least absolute shrinkage and selection operator and multivariate logistic regression. The calibration curves showed excellent agreement between predicted and actual probabilities of INCS insensitivity in both the training and validation sets. The area under the curve values observed in the validation set were 0.918 , and 0.932  in the training set, indicating strong performance on both sets. Decision curve analysis showed that the constructed nomogram yielded a net clinical benefit for AR patients.  The nomogram constructed from risk predictors of INCS insensitivity in patients with AR demonstrated strong predictive power and enabled clinicians to identify high-risk patients, aiding them in developing an optimal treatment plan for AR. Allergic rhinitis (AR) is a type I allergic reaction in which the nasal mucosa becomes inflamed due to an IgE-mediated hypersensitive reaction to certain allergens . The estIntranasal corticosteroids (INCS) are commonly used to treat moderate-to-severe AR, but their effectiveness varies between individuals; however, even with treatment, a patient may not show a significant improvement in their clinical symptoms , 11. INCThrough utilizing demographic and clinical data such as age, gender, length of history of AR, classification of AR, family history of AR, and comorbid conditions, etc., linked to AR, this study created a predictive model to aid medical professionals in determining INCS insensitivity in AR patients.μg taken daily) [This study focused on Chinese individuals aged 18 to 75 years old who had moderate-to-severe AR symptoms that significantly affected their sleep, daily activities, work, or studies  and had n daily) , 16, 17 This study excluded pregnant women, breastfeeding women, individuals who had malignant neoplasms, autoimmune diseases, hepatic or renal diseases, those who had used decongestants in the previous week, those who had taken INCS in the past four weeks, those who had been given systemic corticosteroids in the last eight weeks, those who had been prescribed antileukotrienes or H1 antihistamines in the previous two weeks, those who were undergoing allergen immunotherapy, those who had undergone nasal or sinus surgery, and those who had taken traditional Chinese medicine or acupuncture in the previous two weeks.AR is characterized by recurrent sneezing, runny nose, nasal itching, nasal congestion, and watering eyes, accompanied by physical signs such as paleness, edema, and increased secretions. This diagnosis is confirmed by positive results from skin prick tests (SPT) and/or the detection of elevated serum-specific IgE levels for allergens , 18.This study aims to find predictive factors for insensitivity to INCS within a training dataset, which is used to develop a nomogram. The performance of this nomogram is evaluated by analyzing the data from the training and validation datasets. An overview of the patient screening process and study design is provided in Total nasal symptom scores (TNSS) were recorded before and after the patients used INCS. This survey is used to measure the severity of the main AR symptoms and comprises three questions assessing nasal blockage, itchiness or sneezing, and secretions or a runny nose. Every question is rated on a 4-point scale, with responses ranging from “0” (no symptoms) to “3” (severe symptoms) , 18–20. Duration of AR: The length of the patient's AR is recorded as the interval between the onset of symptoms or diagnosis of AR and their present consultation . AR duraAge: Patients are classified as young (18–44 years old), middle-aged (45–59 years old), or elderly (older than 60) according to their age bracket.Marital status: married, single, or others.Place of residence: Patients are classified as residing in a rural or urban area.The presence or absence of AR sufferers in the patient's immediate family determines if the patient has a positive or negative family history of AR.AR can be divided into two subtypes: intermittent AR, which involves rhinitis symptoms that last for less than four days per week or four consecutive weeks, and persistent AR, which features rhinitis symptoms that last for more than four days per week and four consecutive weeks .INCS use can be classified into two types: first-time users and those who have already experienced using INCS.Comorbidities related to AR can include asthma or others  , 23.https://www.R-project.org) for this study. A two-tailed P value of less than 0.05 was established as the level of statistical significance.Statistical analysis was conducted using the R 4.2.0 software package  regression were applied to select the risk factors. Multivariate logistic regression was used to identify risk factors for INCS insensitivity by examining the associated risk variables. A nomogram was generated using the “rms” package to facilitate the prediction of INCS insensitivity. Calibration plots and Hosmer–Lemeshow (HL) goodness-of-fit tests were used to assess the calibration of the nomogram. Receiver operating characteristic (ROC) curves and areas under the curves (AUC) were calculated for both the training and validation sets to evaluate the accuracy and performance of the nomogram. The clinical utility and net benefit of the predictive models were evaluated using decision curve analysis (DCA).Among the 313 individuals diagnosed with AR, 38.3% were found to be insensitive to INCS therapy. This patient group comprised 64.9% males and 35.1% females. Of these patients, 38.0%, 31.3%, and 30.7% were classified as young, middle-aged, and elderly, respectively. 56.9% of patients had prior exposure to INCS, while the remaining 43.1% were first-time users. λ of 0.06974 and a log (λ) of −2.662981 were chosen, leading to the reduction of nine predictors to four: the type of AR, family history of AR, comorbidities, and duration of AR, as demonstrated in By employing the LASSO technique, The INCS insensitivity nomogram model was created with LASSO regression screening of four variables . To use P = 0.05) in the training cohorts and 2.988 (P = 0.935) in the validation cohorts. In both the training and validation cohorts, the calibration plots of the nomogram demonstrated good agreement between the predicted and observed outcomes. The AUC of the nomogram was measured to be 0.918  in the training dataset and 0.932  in the validation dataset, demonstrating the model's excellent performance. Furthermore, DCA showed that the predictive model had considerable net benefits for the majority of threshold probabilities at various periods of time in the training and validation cohorts, as shown in An analysis of Hosmer–Lemeshow (HL) goodness-of-fit determined that the model's performance was satisfactory, as evidenced by a chi-square value of 18.211 . This composition was similar to a multi-center, large-scale study of people with AR in China , which rAccording to a Chinese study , 23.2% oα (GR-α) or GR-α competitive receptor (GR-β), inadequate interaction between GC and GR or between the GR complex and DNA, reduced GR-α transport, decreased histone deacetylase expression, and/or enhanced activity of pro-inflammatory transcription factors, although there may be some differences.RA patients may be insensitive to INCS due to similar mechanisms as those of fat-soluble glucocorticoid (GC) insensitivity , includiA study focused on the GLCCI1 genotype, rs37973 has demonstrated that AR may be insensitive to glucocorticoids due to its inherited nature . This geα and an increase in GR-β expression, reducing the body's responsiveness to INCS [The prolonged and persistent AR might be associated with an increased insensitivity to INCS. This insensitivity can be attributed to the increase in the exposure of allergens in the environment, which leads to a decrease in the expression of GR- to INCS . Moreove to INCS  found thα transport, as studies have shown that [α in the cells of the respiratory mucosa cannot be translocated to the nucleus, resulting in glucocortical insensitivity.It has been observed that the nasal cavity and bronchi are part of the same respiratory tract and that allergies such as AR and asthma share similar pathophysiology , 20. Conown that , unlike Our study was subject to certain limitations, such as selection bias from participants who decided to discontinue the treatment. We found that approximately 38.3% of the participants were insensitive to INCS. However, it is possible that the number of people insensitive to INCS is higher, as those who are insensitive may not continue taking INCS and therefore were not included in our study. Additionally, due to the limited sample size from one site, more data are necessary to validate the accuracy of the constructed nomogram. Despite the drawbacks, this study presents an interesting and unique, more personalized approach for predicting INCS-insensitive AR. It provides healthcare professionals with a new strategy for treating individuals with AR who are unresponsive to INCS.This study successfully designed and validated a nomogram for predicting INCS insensitivity in adult patients with allergic rhinitis. The nomogram incorporates important risk factors such as type of AR, comorbidities, family history of AR, and duration of AR. The strong performance of the nomogram on both the training and validation sets indicates its potential as a useful clinical tool for identifying patients who may not respond well to INCS treatment. The decision curve analysis also demonstrated that the nomogram can yield a net clinical benefit for AR patients. This nomogram has the potential to improve personalized treatment and management strategies for Chinese adult patients with allergic rhinitis.


Androgen receptor (AR) activation and repression dual-functionality only became known recently and still remains intriguing in prostate cancer (PCa). MYC is a prominent oncogene that functionally entangles with AR signaling in PCa. Further exploration of AR regulatory mechanisms on MYC gene transcription bears clinical and translation significance.Bioinformatics analysis of PCa cell line and clinical RNA-Seq and ChIP-Seq (chromatin immunoprecipitation-sequencing) datasets to anchor interactions of AR and MYC transcriptional networks. ChIP-qPCR and 3C (chromosome conformation capture) analyses to probe MYC distal regulation by AR binding sites (ABSs). CRISPR/Cas9-mediated genome-editing to specify functions of ABS within the 8q24-MYC locus on androgen-mediated MYC transcription. Global FoxA1 and HoxB13 distribution profiling to advance AR transcriptional mechanisms.Here we recognize AR bi-directional transcription mechanisms by exploiting the prominent 8q24-MYC locus conferring androgen hyper-sensitivity. At ~ 25 Kb downstream of the MYC gene, we identified an undefined ABS, P10. By chromatin analyses, we validated androgen-dependent spatial interaction between P10 and MYC-Promoter (MYC-Pro) and temporal epigenetic repression of these MYC-proximal elements. We next designed a CRISPR/Cas9-mediated double genomic knock-out (KO) strategy to show that P10-KO slightly lessened androgen-elicited MYC transrepression in LNCaP-AR cells. In similar genomic editing assays, androgen-mediated MYC repression became slightly deepened upon KO of P11, an ABS in the PVT1 gene locus highly enriched in AR-binding motifs and peaks. We also investigated multiple ABSs in the established PCAT1 super-enhancer that distally interacts with MYC-Pro for transactivation, with each KO pool consistently shown to relieve androgen-elicited MYC repression. In the end, we systemically assessed androgen effects in the 8q24-MYC locus and along PCa genome to generalize H3K27ac and BRD4 re-distribution from pioneer factors (FoxA1 and HoxB13) to AR sites.Together, we reconciled these observations by unifying AR dual-functions that are mechanistically coupled to and equilibrated by co-factor redistribution.The online version contains supplementary material available at 10.1186/s12967-023-04429-4. The advancements in chromatin and epigenetics technologies in recent years have enabled the exposure of AR transrepressive functions that remain elusive for decades –3. In coMYC is a prominent oncogene functionally entangled with AR signaling and its overexpression renders androgen insensitivity and invasive PCa transformation –9. ClustMYC could trigger immediate and reversible cancer-driven signals, making it compelling to specify the regulatory mechanisms of the MYC gene . MYC is Our current report was designated to define the mechanisms of androgen-elicited dual-transcriptional functions in global and local contexts. For this purpose, we focused on the 8q24-MYC locus that contains multiple AR binding sites (ABSs) in annotation with PCa-specific enhancers. Using an innovative CRISPR/Cas9-mediated genome-editing strategy, we deleted several key ABSs from the left and right arms of this TAD, respectively. The outcomes of these KO tests were consistent with the concept that MYC transcription is coupled to AR direct activation and indirect repression functions. In the end, we further took bioinformatics to systemically dissect androgen effects in the 8q24-MYC locus and along the PCa genome, demonstrating that H3K27ac and BRD4 are re-distributed from pioneer factors (FoxA1 and HoxB13) to AR sites. Collectively, our findings recognized AR-mediated transcription is intrinsically equilibrated between its dual-functions that are coupled to co-factor exchanges.DHT , G418 , Puromycin , Hygromycin B , Blasticidin , Lenti-gRNA-Neo (G418) vector  and Lenti-gRNA-Puro vector . Transfections were conducted using Lipofectamine 2000 , following the manufacturer’s direction.AR , BRD4 , H3K27ac , H3K4me1 , H3K4me2 , H3K9me1 , H3K9me2 , H3ac , FoxA1 , Med1 , CDK9 , cyclin T1 , p-RNA Pol II-Ser5 , and IgG .LNCaP cells were grown in RPMI medium containing 10% FBS and VCaP cells were grown in DMEM medium containing 10% FBS. LNCaP AR overexpression (OE) stable line (LNCaP-AR or LA) was generated with lentiviral vector expressing Flag-tagged AR and hygromycin B selection; LNCaP-AR Cas9 OE stable line (LNCaP-AR-Cas9 or LAC) was generated based on lentiviral vector expressing Cas9, under Hygromycin B and Blasticidin selection . For CRIThe procedure was reported . BrieflyThe detailed information was included in Additional file The test was conducted using the Fast Virus 1-step kit . The TaqMan primer–probe sets were all from Life Technologies (Applied Biosystems). Primer/FAM-probe sets: MYC (Hs00153408_m1), PSA/KLK3 (Hs02576345_m1), PVT1 (Hs01069041_m1); and the CCAT1 gene TaqMan primer/FAM-probe set: forward primer: GGCCAGCCCTGCCACT; reverse primer: CAGTTTTCAAGGGATTTTAGGAGAA; and probe: ACCAGGTTGGCTCTGTATGGCTAAGCGT. The inventoried internal control is the GAPDH primer/VIC-probe set (4310884E).ChIP-Seq and RNA-Seq datasets used in this study are retrieved from the GEO database, as listed below: LNCaP AR ChIP-Seq, GSE83860 ; LNCaP HRaw ChIP-Seq data were trimmed and then mapped to either Hg19 or Hg38 reference genome as indicated. MACS2  and ucscGenerally, data in bar graphs represent mean ± SD of three biological replicates and are representative of three independent experiments. Two-tailed unpaired Student’s t-test was performed to calculate the statistical significance of two independent groups. P < 0.05 was considered statistically significant: *P < 0.05, **P < 0.01, ***P < 0.001.Recent advancements on chromatin techniques revealed the bi-directionality of AR-mediated transcription: transactivation versus transrepression –3. ConsiThe prominent 8q24-MYC locus is a gene desert featured in residency of MYC and flanking lncRNAs and multiple prostate risk regions , 20. MecMYC is known to have no proximal enhancers and depends on distal elements for transactivation , 19, andTo further expose the mechanisms of androgen-mediated MYC transrepression, we monitored epigenetics of the MYC-Pro and P10 sites in DHT-treated VCaP with ChIP-qPCR analyses. At both sites, androgen stimulated AR and FoxA1 occupancy, enhanced the transrepressive markers H3K9me1 and H3K9me2, and repressed the transactive markers H3K4me1 and H3K4me2  under G418 selection and a downstream gRNA pair (D1/D2) under Puro selection, respectively . As shown in Fig. We next similarly targeted an ABS P11) in the MYC 3′ flanking lncRNA PVT1 region that is enriched in AR binding motifs (ABMs) Fig. A. Consis1 in the The control versus KO pools in androgen-depleted medium were then subjected to DHT treatment as indicated, followed by TaqMan RT-PCR assay for MYC expression Fig. B–E. As sPrevious report established the PCAT1 SE as a robust developmental and locus-wide enhancer that distally engaged with MYC-Pro , 20. We As aforementioned, co-factor re-distribution intrinsically couples with androgen-elicited transcriptional activation and repression. Specifically, each individual androgen-elicited gene and chromatin site exhibited distinct responsive profiling; for example, the magnitude and duration of androgen responsiveness are different among genes in the left arm, middle section, and right arm of the 8q24-MYC locus [To generalize our findings along the PCa genome, we next took global bioinformatics strategy on VCaP datasets. For this purpose, we focused on FoxA1 and HoxB13, two key pioneering factors in PCa AR reprogramming , 32. By The above findings supported that androgen elicited indirect repressive effects by transferring co-activators from FoxA1 and HoxB13 sites to AR sites. To affirm these findings, we next systemically addressed chromatin occupancy of these factors at AR-binding sites along the VCaP genome. As shown, DHT impacted negatively basal-AR binding affinity but positively liganded-AR binding affinity; similar trends were noted on H3K27ac binding affinity at these AR sites, consistent with re-distribution of H3K27ac from androgen-repressed to androgen-activated AR sites  PCa cell lines. As shown in the androgen-refractory LNCaP-abl cells, FoxA1 KD by siRNA had repressive effects on ABS enhancers (particularly the PCAT1 SE) on left arm of this locus and MYC transcription  PCa cells impacted 8q24-MYC gene locus activities and MYC gene expression. Additional information on materials and methods