To pursue high quality research, successful participant recruitment is essential, but recruitment rates are often low. This is specifically true in target populations with impairments, for instance, among stroke survivors. Previous studies focusing on recruitment have mainly relied on information from professionals, and there is therefore a need to contribute with new methodological insights to how potential rehabilitation research participants describe their interest and preferences to participate in research. The purpose of this study was to generate knowledge about stroke survivors’ interest in participating in rehabilitation research, reasons for being interested or not, and preferred forms and foci of rehabilitation interventions. An additional aim was to describe preferences regarding survey administration modes and processes for recruitment to studies.This cross-sectional study recruited Swedish residents who had sustained a stroke, initially by using advertisement on the National Stroke Association’s website, flyers posted at local occupational and physical therapy offices and at local stroke/senior organization meetings. Secondly, participants were recruited through a local stroke register. The survey, administered either in a paper form returned by postal mail; online or as a phone interview with 128 stroke survivors.p = 0.001) and those closer to stroke onset (p = 0.047). Contribution to research, possibility to try new rehabilitation interventions and meeting others in the same situation were reasons that attracted an interest to participate. Other important aspects were related to motivation, individual needs, as well as how skilled the people who provided the intervention were. Participants preferred group-based programs, and programs focusing on regaining lost functions were highly requested. A majority wanted to be contacted through postal mail (70%) and most of them (90%) used the paper form to respond to the survey.Most of the participants were interested in participating in rehabilitation research, particularly younger persons (A range of personal and external aspects, including challenges related to digitized administration modes, should be considered to achieve high participation rates in rehabilitation research targeting stroke survivors. The importance of addressing individual needs and prerequisites in an individualized manner should not be underestimated and might be a useful strategy to recruitment success. To pursue high quality research, successful participant recruitment is essential but response rates are often low in health-related surveys  and randRecruitment to rehabilitation research is particularly exposed to risk of selection bias as the target populations are people with impairments , for exaChallenges to study participant recruitment are related to individual and societal expectations, for example, stroke survivors’ lack of interest and transport possibilities, health issues , 8, old The purpose of this study was to generate knowledge about stroke survivors’ interest in participating in rehabilitation research, reasons for being interested or not, and preferred forms and foci of rehabilitation interventions. An additional aim was to describe preferences regarding survey administration modes and processes for recruitment to studies.We designed a cross-sectional study with data collection based on a self-administered survey, created and piloted for the purposes of the present study, utilizing different recruitment methods.We recruited people who had sustained at least one stroke event in adulthood (≥ 18 years), were resident in Sweden and able to independently or through support from another person fill in a self-administered survey in Swedish. Multiple recruitment modes were used. As a first round of recruitment, we used the two following methods; i) advertisement on the National Stroke Association’s website, and ii) flyers posted at local occupational and physical therapy offices, at local stroke organization member meetings and senior organization meetings. Due to the considerable difficulties experienced while using the two modes, direct postal surveys were sent to the stroke survivors on the local Lund Stroke Register (LSR)  to facilA self-administered two-part questionnaire constituting the survey was constructed in an interactive process by the co-authors who had different professional/disciplinary backgrounds and expertise. The first part was study-specific and included 18 questions. In addition to demographic questions , the questionnaire captured interest in rehabilitation interventions  and in participation in rehabilitation research, reasons for the interest, contact mode preferences, as well as preferred intervention forms and foci. The latter questions had a multiple-choice structure, with the possibility for more than one response option. In addition, the open-ended question “What do you think is important for researchers to consider when recruiting stroke survivors’ to rehabilitation research?” was included. Prior to the data collection, the questionnaire was piloted with five stroke survivors (not included in the study sample). Only minor wordings were altered after the discussion.The second part included questions from three established questionnaires: The Stroke Impact Scale, 2.0 (SIS) ; the GenThe numerical data from the survey were analyzed using descriptive statistics, and the qualitative data from the open-ended question were analyzed through thematic analysis .p < 0.05. The SSPS Statistics 22.0 software  was used to analyze the data.To improve the interpretability of the results the “interested in participating in rehabilitation research” variable was dichotomized. Comparisons between interested/not-interested participants were computed to identify differences in relation to age, gender, time since stroke, SIS domains, GSE and GDS. Mann-Whitney U or Chi-square tests were applied as appropriate. Missing values were imputed using documented rules for SIS, GSE , 22 and A total of 128 participants (52 women and 41 men) with a median age of 73 years were included in the study. The median time since stroke onset was 1 year or more for over 90% of the participants. Most of them lived with a partner (66%), in a single-family house (54%), and in an urban area (86%). Participant characteristics are further described in Table N = 128). The data collection were performed prior to the covid-pandemic. Among the 128 people who answered the survey, the response rates for each recruitment mode were 90% (advertisement), 37% (flyers/presentations at meetings) and 30% (patient register). There were no significant differences in response mode across gender, but age differed significantly between the advertisement and patient register modes . In addition to the 128 responses, 10 questionnaires were resent blank with a note, or a family member phoned to explain why no response was made . The vast majority delivered their completed survey in paper form (90%). There were a few online (9%) and phone (1%) responses. No differences in administration mode were observed across gender (p = 0.906), age (p = 0.062) or recruitment mode (p = 0.110).In total, 332 surveys were distributed, resulting in a 39% response rate ’ had a median of 7.n = 105; 82%) indicated that they were interested in participating in rehabilitation research. More than 50% (n = 69) replied “definitely interested” and about a third “maybe” interested. The remaining (less than a fifth) answered “definitely not interested”. Younger participants were more interested than older (p = 0.001) and there was a lower interest among those who had their stroke onset a longer time ago, compared to those who recently had their stroke (p = 0.047). There were no statistically significant differences between those interested in participating in rehabilitation research and those that were not across each of the participants’ gender, strength, cognitive impact, general self-efficacy or possible depression, see Table A majority of the participants (The three most reported reasons for participants’ interest in rehabilitation research were to contribute to research (69%), try new rehabilitation programs 48%), and meet others in the same situation (46%) ! Improvements can occur even many years after the stroke”.If invited to participate in rehabilitation research, the most preferred information channels were through postal mail (70%), personal meetings (36%) or phone calls (34%). Advertisement through the Internet  was less attractive (21%), and so were newspaper (3%) and TV/radio (3%). Sixty-three (49%) responded to the open-ended questions, and indicated that information initially should be directed to family members, and that programs should be conducted not long after the stroke event: More than two thirds (67%) expressed a high interest for participating in group-based interventions conducted in, for example, primary care, hospital or university facilities. This was also reflected in the responses to the open-ended questions. Almost one-third (29%) were interested in interventions where the people who provided the intervention made home visits, and 19% in interventions delivered by phone. Interventions combining home visits and phone calls attracted 13%, Internet/video based interventions 14% if performed individually, and 3% if performed in a group format.Rehabilitation research focusing on regaining lost physical or cognitive functions were highly requested (85%), followed by research on interventions focusing on learning strategies to manage tasks and activities that can be difficult to perform after stroke (59%). Interventions focusing on finding new/alternative ways of performing daily activities (including provision of assistive devices/housing adaptations) were of interest for a good third (38%).“everyday life” and “psychological stress”. A recommendation regarding the form of the rehabilitation interventions was to offer interventions to similar target groups that shared similar impairments or problems: “Coordination can be done with other neurological diseases, for example, Parkinson’s”. The participants also stated a need of adapting rehabilitation to participants with different types and levels of impairments, bearing in mind their age and life circumstances: “An important thing is to see the difference in age. What I, a woman not even 40 with small kids, need compared to somebody newly retired or in an age of 80 needs when it comes to rehabilitation should differ in some respects. Not the least cognitively. … Most people who get strokes are old and this causes trouble for us younger. This must be taken into consideration, so we can get help as well”. Furthermore, some participants highlighted individual incentives that could encourage participation in rehabilitation research. They indicated that those who have actual needs of rehabilitation may be more positive than others.One theme that emerged from the open-ended responses was the importance of taking a broad spectrum of individual needs into account to promote participation in rehabilitation research. It was related to the foci of rehabilitation interventions of interest such as “That they have an “open mind” and want to improve their situation although the years have past” and “That they can and dare to formulate their thoughts”. Lastly, they stressed that the approach and competence of those who provided the intervention are essential, reflected by statements like “Kindness and patience” and “Do not hurry”.Another theme focused on personal characteristics of participants and those who provided the interventions that would promote participation in rehabilitation research. Genuine interest and motivation among the potential participants as well as confidence and ability to communicate were mentioned: In this study, we embraced a user perspective and explored opportunities to recruit stroke survivors as participants in rehabilitation research, which is an urgent methodological challenge for many researchers . This stThe answers to the open-ended question stress that it is important that the content of rehabilitation research consider a broad spectrum of needs, not only type of disability, but also age and life situation. For instance, some of the younger participants in our sample stressed that their needs were not taken into account because they were put together with older persons who had other expectations and needs. This may reflect how local rehabilitation services in general are organized, and carried out. For example, where persons with the same diagnosis risk receiving the same rehabilitation without consideration for individual needs based on person-centered care . FurtherAlthough earlier research has documented that there are several challenging factors regarding recruitment to stroke research –9, 11, aNot only is the format of rehabilitation interventions important, but also their foci. Our study highlights that many stroke survivors prioritize rehabilitation research focusing on regaining lost physical or cognitive functions. This is presumably associated with a strong hope for recovery  and not A specific result worth emphasizing is that a rather high proportion of our participants mentioned that obtaining support to travel to where the rehabilitation takes place is a key issue. Many stroke survivors cease to drive a car  and findThe importance of the competence of the investigators and those who provide the intervention in rehabilitation research cannot be overestimated, especially in terms of their skills. Carlstedt et al.  assertedMoreover, to promote study participation in rehabilitation research, aspects such as participant interest and motivation are important. Evaluations of readiness for making behavioral changes according to Prochaska’s and DiClemente’s change model  might beOne limitation and weakness of the present study is the unknown overall response rate, which as such is related to the study rationale. As this study was initiated based on the experiences from a previous study, it ended up with a very low response rate despite major efforts to recruit a reasonable sample (26). Therefore, the present study relied on a sampling procedure striving to utilize several complementary strategies, and the weaknesses are a direct consequence of the recruitment procedure. Moreover, the included subjects are likely biased towards being stroke survivors willing to participate in rehabilitation research. Despite major efforts, we were unable to achieve a high response rate and recruit a larger sample. This is reflective of the challenges in obtaining responses to surveys , emphasiFurthermore, the composition of the study sample should be mentioned. The prevalence of cognitive impairments in our study was low compared to in the general population of stroke survivors . On the A strength of the present study is that we collected data from potential research participants’ perspective, and not from professionals. We utilized several recruitment and administration modes, and had generous inclusion criteria to reach a broad sample of stroke survivors. We used quantitative and qualitative data collection, making it possible to answer the research questions from different perspectives in a complementary manner. The finding that the vast majority of the responding stroke survivors prefer to be contacted by postal letter and use paper forms to respond speaks for consciousness in this regard when using surveys with this specific population. In an international perspective, it is important to keep in mind that Sweden is among the most digitalized countries in the world  and incrAlthough recruitment of stroke survivors to rehabilitation research is a challenging task, there are study participants who are highly interested. Recommending potentially successful methodological strategies based on the results of the present study, a range of personal and external aspects should be taken into account to achieve high participation rates in rehabilitation research targeting stroke survivors. The importance of addressing needs and prerequisites in an individualized manner should not be underestimated and might be a useful strategy to recruitment success. Although the study sample was small and presumably biased towards stroke survivors who already had an interest to participate in rehabilitation research, the results can be used to guide researchers when designing and planning for new projects. A noteworthy methodological implication is that although we offered several administrative modes, the majority used the traditional paper form and wanted to be contacted through postal mail. This is contradictory to the ongoing digitalization of society and needs to be further explored.


AC016745.3 in the development of PCa. It shows that dihydrotestosterone (DHT) results in the AR-dependent suppression of AC016745.3 expression in the LNCaP androgen-sensitive human prostate adenocarcinoma cell line. In addition, overexpression of AC016745.3 inhibits the proliferation and migration of PCa cells, and suppresses the expression of AR target genes. This research also demonstrates that the protein NONO interacts with AR and functions as an AR co-activator, promoting AR transcriptional activity. Furthermore, using RNA immunoprecipitation (RIP)-PCR experiments, the study demonstrates that both NONO and AR can bind AC016745.3. Moreover, cell phenotypic experiments reveal that NONO can promote cellular proliferation and migration, and that AC016745.3 can partially antagonize the pro-oncogenic functions of NONO in PCa cells. In summary, the results indicate that AC016745.3 can bind NONO, suppressing its ability to promote AR-dependent transcriptional activity. Furthermore, DHT-dependent suppression of AC016745.3 expression can enhance NONO’s promotion effect on AR.The androgen receptor (AR) and its related signaling pathways play an important role in the development of prostate cancer (PCa). Long non-coding RNAs (lncRNAs) are involved in the regulation of tumorigenesis and development, but their specific mechanism of action remains unclear. This study examines the function and mechanisms of action of lncRNA  Prostate cancer (PCa) is a common malignancy of the male reproductive system. The incidence of prostate cancer ranks second among male malignancies worldwide . The andPlncRNA-1, HOTAIR, and PRNCR1, can both interact with and affect the transcriptional activity of AR [Long non-coding RNAs (lncRNAs) are defined as transcripts of more than 200 nucleotides that are not translated into proteins . There ity of AR ,13,14,15AC016745.3 as an androgen-responsive lncRNA of unknown function that is differentially expressed in PCa. AC016745.3 is 508 bp in length and is located within an intergenic region. This current study shows that AC016745.3 can function as a tumor-suppressor in PCa, and that increased AC016745.3 expression inhibits AR-dependent gene transcription in LNCaP cells. The results indicate that AC016745.3 interacts with the AR co-activator NONO, suppressing its transcription-promoting activity towards AR. Conversely, decreased expression of AC016745.3 in response to dihydrotestosterone (DHT) stimulation can enhance NONO’s promotion effect on AR.The authors of this paper recently identified 2.LNCaP cells were purchased from the American Type Culture Collection  which were confirmed by short tandem repeat analysis. DU145 and PC-3 were obtained from Cell Bank of Chinese Academy of Sciences  and were authenticated by mycoplasma detection, DNA-fingerprinting, isozyme detection, and cell vitality detection. LNCaP, PC-3, 22Rv1 and DU145 were cultured routinely using RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA), 1% sodium pyruvate (PS), and 1% ampicillin/streptomycin sulfate (S/P). All cells were cultured at 37 °C in 5% CODehormone starvation treatment was undertaken using RPMI 1640 phenol red-free medium with 5% activated carbon adsorption-treated fetal calf serum (CCS-FBS), 1% non-essential amino acids (NEAA), 1% sodium pyruvate (PS), and 1% ampicillin/Streptomycin sulfate antibody (S/P). LNCaP cells treated with androgen after three days of hormone starvation treatment. According to the experimental requirements, the DHT treatment dose was 0.1 nM-1000 nM final concentration, the treatment time was 2–48 h, and the corresponding volume of ethanol was used as a control.All small interfering RNA (siRNA) oligonucleotides against NONO and negative control (NC) were purchased from GenePharma , and used at 50 nM concentration. See −ΔΔCT method. Refer to RNA was purified with TRIzol (Invitrogen) and reverse transcribed with PrimeScript RT Master Mix (Takara) using 500 ng of RNA and 0.25 µM of random hexamers. qPCR was performed using the SYBR Green PCR Master Mix (Vazyme). The data were analyzed using the 27 cells were lysed with the prepared NP40  to extract the nuclear proteins. The nuclear proteins were mixed with a concentration of 2 mg/mL and 50 pmol Biotin-Labeled RNA in buffer NT-2  and incubated for 2 h, then 60 μL of Streptavidin agarose  was added, and incubated for 1 h. After the beads are eluted with buffer NT-2, the target protein was identified by mass spectrometry or Western blotting analysis.The T7 promoter-containing DNA fragment was used as a template for in vitro transcription according to the product instructions  and followed by RNA extraction . For labAn Orbitrap mass spectrometer (Thermo Fisher) was used to perform mass-spectrometric sequencing with RNA pull-down protein. The sample was loaded onto a CAPTRAP column  in 5 min at a flow rate of 10 μL/min. The sample was subsequently separated by a C18 reverse-phase column  at a flow rate of 300 nl/min. Protein searches were performed with Mascot 2.3.02 software (MatrixScience) against the SWISSPROT human protein database (release 2014_07). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner 7 cells were lysed with 0.5% NP40 and the supernatant was collected as a lysate by centrifugation. An amount of 50 μL of Protein-A magnetic beads was equilibrated with 5% BSA-NT2 buffer and 3–5 μg of antibody was added, then incubated overnight at 4 °C. The beads were washed 4–5 times with pre-chilled NT2 buffer, and cell lysate added and mixed well. An amount of 100 μL was taken as Input, and the remaining samples were incubated at 4 °C for 4 h. The magnetic beads were washed 4 times with pre-chilled NT2 buffer. An amount of 500 μL Trizol was added to extract RNA for qRT-PCR analysis. The following antibodies were used: anti-AR , anti-NONO  and anti-IgG .An amount of 2 × 10Chromatin immunoprecipitation (ChIP) was performed as described previously . Chromat7 cells were lysed with 0.1% NP40 and the supernatant was collected by centrifugation. The AR antibody added to the cell lysate, and incubated overnight at 4 °C on a rotating wheel. Protein A agarose beads  or protein G agarose beads  were added to the lysate and spun at 4 °C for 4 h. The centrifuge was set at 2000 rpm for 3 min, the supernatant was discarded to collect the precipitate, and washed 4 times with Wash Buffer. The supernatant buffer was added and then boiled for 10 min, and centrifuged to collect the supernatant for mass spectrometry or Western blotting analysis.An amount of 1 × 10An amount of 10,000 digested cells were seeded in the upper layer of each transwell chamber of a 24-well plate, and cultured for 48–72 h. Cells were fixed with 100% methanol, and migrating cells were stained with DAPI staining solution. The number of cells that migrated to the lower layer of the chamber were observed with an Olympus inverted fluorescence microscope.A cell proliferation assay was performed as described previously using Cell Counting Kit-8  according to the manufacturer’s instructions. Absorbance was measured at 450 nm with Microplate Reader ELx808  at different time points, the absorbance at 630 nm was used as a reference.Cells were collected and treated with 0.03% Triton X-100 and 50 ng/mL propidium iodide (PI) for 15 min in the dark. The percentage of cells in different cell cycle phases  were measured by FACScalibur flow cytometer .Cell apoptosis was determined with the FITC-Annexin V Apoptosis Detection Kit  by FACSCalibur flow cytometer (BD) according to the manufacturer’s instructions.p < 0.05 was considered statistically significant with a 95% confidence level.The numerical data were presented as mean ± standard deviation (SD) of at least three determinations. Statistical comparisons between groups of normalized data were performed using T-test or Mann–Whitney U test according to the test condition. AC016745.3 as an androgen-responsive RNA, LNCaP cells were treated with the androgen DHT and changes in AC016745.3 expression were examined. These experiments revealed that DHT treatment resulted in both a dose-dependent and time-dependent decrease in the expression of AC016745.3  within the AC016745.3 gene was undertaken using Genomatix software, and two putative sites were identified . qRT-PCR experiments show that lncRNA AC016745.3 was present in both the nucleus and the cytoplasm following initial DHT stimulation, but by 2 h its expression was predominantly restricted to the nucleus  experiments were next performed to test whether AC016745.3 is present in a complex with AR. These experiments revealed that, following DHT treatment, AC016745.3 was significantly enriched in AR immunoprecipitates in LNCaP cells, indicating this lncRNA may have interacted with AR or AR complex in LNCaP cells , and partially derepress the expression of genes negatively regulated by AR , following cell treatment with DHT for 2 h , 759 proteins were identified that interacted with AR , and inhibited the expression of SREBP-1a, a gene negatively regulated by NONO , a finding that is in agreement with NONO’s role as an AR cofactor  protein family, and has been shown to be overexpressed in PCa [AC016745.3 were screened out, including NONO. NONO possesses conserved N-terminal RNA recognition motifs and a DNA binding domain [AC016745.3 interacts with NONO, thereby affecting the transcriptional activity of AR.The formation of an AR transcriptional complex requires the functional and structural interaction of the AR with its coregulators. Recent studies have shown that some lncRNAs can interact with and influence the transcriptional activity of AR ,13,14,15d in PCa ,26. Althd in PCa ,28, its g domain ,30. NONOAC016745.3 and AR increases significantly following DHT stimulation, but that NONO binds AC016745.3 independently of DHT. This finding demonstrates that, under conditions of DHT stimulation, AC016745.3 binds to the AR transcription complex following its recruitment by NONO [AC016745.3 antagonizes the activity of NONO. It was also found that AC016745.3 expression was reduced under conditions of DHT stimulation, and that this impaired the antagonistic effects of AC016745.3 towards NONO. Through cell phenotype experiments, it was found that AC016745.3 can partially restore the cancer-promoting properties of NONO in PCa cells, suggesting that AC016745.3 and NONO may antagonize one another in PCa.Ishitani et al. have reported that DHT can promote the interaction between NONO and AR . In the  by NONO . ConsequAC016745.3 only partially restores the function of NONO in PCa cells suggests that the cancer-promoting properties of NONO are only partly driven by AR activity, and that other AR/AC016745.3-independent mechanisms must also exist. Emerging evidence shows that NONO engages in almost every step of gene regulation, including, but not limited to, mRNA splicing, DNA unwinding, transcriptional regulation, nuclear retention of defective RNA, and DNA repair [The observation that A repair . As suchAC016745.3 and NONO still play a role in AR-negative prostate cancer cells. Our understanding is, that in addition to interacting with AR to affect the transcriptional activity of AR, NONO can also perform transcription activity in an AR-independent manner, and the interaction between AC016745.3 and NONO does not depend on AR, therefore, they also play a role in AR-negative prostate cancer cells, but this is not the focus of this article. This work deserves further study in the future.These functional experiments found that AC016745.3 on PCa apoptosis should be further validated using more assays. Secondly, the complicated regulatory relationship among AR/AC016745.3/NONO and other coregulators should be further confirmed, which will strengthen the findings of this study. Finally, while NONO has been confirmed to interact with AC016745.3, its detailed role in PCa remains largely unclear.Several limitations of this study should also be noted. Firstly, the effect of AC016745.3 negatively regulates the transcriptional activity of AR by binding to NONO. Following DHT stimulation, AR negatively regulates the expression of AC016745.3, thereby forming a negative feedback loop, which ultimately enhances AR activity and promotes tumorigenesis.In conclusion, the following working model is proposed based on the results of this study E: NONO i


TMPRSS2 shows negative correlation with the oncogenic events in LUAD. RNA-seq data show that NRP1 level is slightly decreased in peripheral blood of ICU admitted COVID-19 patients, unaltered in lung, while TMPRSS2 level is significantly decreased in lung of COVID-19 patients. Our analysis suggests NRP1 as a potential therapeutic target, while sets an alert on targeting TMPRSS2 for treating comorbidity of COVID-19 and lung cancers.The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), and is highly contagious and pathogenic. TMPRSS2 and Neuropilin-1, the key components that facilitate SARS-CoV-2 infection, are potential targets for treatment of COVID-19. Here we performed a comprehensive analysis on NRP1 and TMPRSS2 in lung to provide information for treating comorbidity of COVID-19 with lung cancer. NRP1 is widely expressed across all the human tissues while TMPRSS2 is expressed in a restricted pattern. High level of NRP1 associates with worse prognosis in multiple cancers, while high level of TMPRSS2 is associated with better survival of Lung Adenocarcinoma (LUAD). Moreover, NRP1 positively correlates with the oncogenic Cancer Associated Fibroblast (CAF), macrophage and endothelial cells infiltration, negatively correlates with infiltration of CD8 Lung cancers, the most commonly diagnosed cancer in males and third in females, account for the highest number of cancer-related deaths in combined sexes globally . The cor+ T cells reinvigoration in response to checkpoint inhibitors [NRP1 is a highly conserved single-pass plasma membrane receptor, which binds with Semaphorins and VEGF. Because of the interaction with VEGF, NRP1 enhances angiogenesis, including the tumor angiogenesis. NRP1 is found to be upregulated in several types of cancer and favors tumor cell growth, survival and metastasis . In addihibitors . DespiteTMPRSS2 codes for a transmembrane serine protease, which can stimulate a proteolytic cascade to promote degradation of extracellular matrix and the cell invasion of prostate cancer . MoreoveACE2, angiotensin-converting enzyme 2, converts angiotensin II to angiotensin-(1–7), which induces the release of vasodilators and plays a protective role in cardiovascular disease and diabetes . Moreovein silico analysis on NRP1, TMPRSS2 in LUSC and LUAD with online databases. NRP1 is widely expressed across multiple organs, while TMPRSS2 is expressed in a restricted pattern. Both are decreased in human lung cancer tissues, consistent with the increased methylation in promoter area and increased microRNAs that target NRP1 or TMPRSS2 in lung cancer tissues. LUSC patients with high NRP1 level have poor prognosis, while LUAD patients with high TMPRSS2 level show better survival rate. Consistent with the association between gene abundance and survival curve, NRP1 is positively correlated with pro-tumorigenic immune cells and genes in cancer tissues in LUSC, while TMPRSS2 shows no correlation or negatively associated with the factors that may facilitate cancer progression in LUAD. Finally, the expression level of NRP1 and TMPRSS2 in COVID-19 patients was analyzed using the public datasets: NRP1 is decreased in peripheral blood of COVID-19 patients admitted to intensive care unit (ICU), but the level of NRP1 in the lung of COVID-19 patients is not changed. TMPRSS2 is significantly decreased in the lung of COVID-19 patients. Taken altogether, NRP1 might be a potential target for comorbidity of COVID-19 and lung cancer, although further experimental validation is lacking. However, TMPRSS2 should not be targeted in lung cancer patients infected with SARS-CoV-2.In this study, we conducted a systemic Tissue-specific expression profiling of NRP1, TMPRSS2 and ACE2 were analyzed through online database, the Human Protein Atlas, which provides expression information based on immunohistochemistry and high-throughput mRNA sequencing . In contACE2 expression level is hardly detective in peripheral blood or lung compared with NRP1 and TMPRSS2 , 1B, andIn the mammalian genome, DNA methylation is an epigenetic modification by which methyl groups are added to DNA, resulting in gene transcription repression . DNA metMicroRNA, a 19-22 nucleotide small non-coding RNA, can bind to the 3’UTR of targeted mRNA which leads to a mRNA degradation or translation inhibition . hsa-mirIn attempts to gain better insights into the role of NRP1 and TMPRSS1 in cancers, the survival curve of cancer patients with differential expression level of NRP1 or TMPRSS2 was analyzed with the Kalpan-Meier plotter, a large database combining information from GEO, EGA and TCGA. High NRP1 expression correlates with reduced overall survival for sarcoma (SARC) , CESC F, TesticuConsistent with the result that low level of NRP1 associates with better prognosis, high level of miR-148a associates with better clinical outcome in multiple cancers, including LUAD , Liver HImmune cell infiltration in tumors correlates with the development of cancers and is of prognostic value in multiple types of tumors, including lung cancers –25. To t+ and CD4+ T cells are cytotoxic immune cells that directly or indirectly kill cancer cells [+ T cell  in LUSC  and CD4+ T cell  in LUAD (Cancer associated fibroblast (CAF), Macrophage and Endothelial cells are usually considered to be pro-tumorigenic, favoring tumor angiogenesis, metastasis and chemotherapy resistance, although they demonstrate great heterogeneity and diverse functions –29. NRP1er cells , 31. NPR in LUSC , while T in LUAD . Consist in LUAD . The rol in LUAD , while T in LUAD .To gain insights into the mechanism of high NRP1 level and low TMPRSS2 level associating with worse prognosis in lung cancers, specific molecules were analyzed to further dissect the immune infiltration correlating with the expression of NRP1 or TMPRSS2. M2 macrophage polarization usually promotes tumor angiogenesis, growth and metastasis , 34. TIMMatrix metalloproteinases (MMPs) are a group of endopeptidases that can degrade extracellular matrix to facilitate tumor invasion and are also involved in angiogenesis to promote cancer cell growth and migration . VEGFC ain vivo, it is necessary to understand their expression kinetics after SARS-CoV-2 infection. Online public data (GSE157103 and GSE147507) of RNA-seq with peripheral blood and lung samples from COVID-19 patients and health controls were subjected to analysis. Results show that the expression of NRP1 is decreased in peripheral blood of COVID-19 patients who are admitted into intensive care unit (ICU), but not in non-ICU COVID-19 patients . Expression level of NRP1, TMPRSS2 in different tumors and paired controls is analyzed with GEPIA (http://gepia.cancer-pku.cn/) and Diff Exp module in TIMER database (https://cistrome.shinyapps.io/timer/). Expression level of hsa-miR-148a-3p, hsa-miR-98-5p is analyzed with the Pan-Cancer module in ENCORI [http://starbase.sysu.edu.cn/index.php), which integrates expression profiles of both non-coding RNA and all protein-coding genes from TCGA.Expression level of ACE2, NRP1 and TMPRSS2 in different tissues was analyzed with The Human Protein Atlas   and GEPIA database. Survival curve of lung cancer patients with differential level of hsa-miR-148a-3p and hsa-miR-98-5p is analyzed with Kaplan-Meier Plotter and Pan-Cancer module of ENCORI.Survival curve of cancer patients with differential level of NRP1, TMPRSS2, is analyzed with the Kaplan-Meier Plotter  (https:/http://explorer.cancerregulome.org/). Pairwise correlation was analyzed with the Spearman’s rank order correlation. Only the genes with -log (p)≥ 6 were shown in the plot.The expression of NRP1, TMPRSS2 and its correlation with other genes in tumors were integratively visualized as a circus plot using the web tool Cancer Regulome .The correlation between NRP1, TMPRSS2 expression and the infiltration level of immune cells or the levels of several pro-tumorigenic genes were analyzed with Gene module in TIMER web server . 1μg RNA was reverse transcribed into cDNA with the PrimeScript® RT reagent Kit with gDNA Eraser . cDNA was 100-times diluted and RT-PCR reactions were then performed in triplicates using TB Green Premix Ex Taq II . Primer sequence is as follows:hGAPDH-F: GTCTCCTCTGACTTCAACAGCG;hGAPDH-R: ACCACCCTGTTGCTGTAGCCAA;hNRP1-F: AACAACGGCTCGGACTGGAAGA;hNRP1-R: GGTAGATCCTGATGAATCGCGTG;hTMPRSS2-F: CCTCTAACTGGTGTGATGGCGT;hTMPRSS2-R: TGCCAGGACTTCCTCTGAGATG;U6-Control-F: CTCGCTTCGGCAGCACAU6-Control-R: AACGCTTCACGAATTTGCGThsa_pre_mir98_F: TTCTGCTCATGCCAGGGTGhsa_pre_mir98_R: ACCAGGGAAAGTAGTAAGTTGhsa_pre_mir148a_F: GAGGCAAAGTTCTGAGACAChsa_pre_mir148a_R: GTTCTGTAGTGCACTGACGraphs were generated with GraphPad PRISM software. Statistical significance was calculated in R version 3.2.3 by Student’s t test. The null hypothesis of the medians/means being equal was rejected at α = 0.05 and p values were generated by unpaired Student’s t test and presented in figures.Supplementary Figures


MAGE family, UXT and FLNA, presented variable methylation levels, but were mainly hypomethylated and therefore all transcriptionally active. The results of this study indicate that CADs present AR monosomy, paralleled by higher transcriptional activity of the gene with potential to influence response to AR deprivation therapy.Carcinomas with apocrine differentiation (CAD) of the breast are rare tumours typically presenting high immunohistochemical expression of androgen receptor (AR) which is a target molecule for personalised therapy. To date, no studies have evaluated the genetic changes that are associated with AR immunohistochemical expression in CADs. The present work aims to characterise AR status in CADs. Twenty CAD tumours were studied with immunohistochemistry, in situ fluorescence hybridization and DNA methylation analysis, to evaluate AR expression and its regulator status. All tumours demonstrated high AR immunohistochemical expression, with over 95% of the neoplastic cells showing AR positivity in 19/20 cases. CADs showed AR gene copy loss in a percentage of neoplastic cells ranging from 5 to 84% (mean 48.93%). AR regulator genes, including the The online version contains supplementary material available at 10.1007/s00428-021-03028-2. HER2 amplification, in addition to strong and diffuse AR positivity on immunohistochemistry [In the study of oestrogen (ER) and progesterone (PR) receptor-negative tumours, attention is currently focused on cases that express androgen receptor (AR). In most of the cases, these tumours display the morphological features of carcinoma with apocrine differentiation (CAD) of the breast 1), composed of large cells with abundant, granular and eosinophilic cytoplasm, a centrally located nucleus with a thick nuclear membrane, coarse nuclear chromatin and a prominent nucleolus , compose. Gross chemistry . AR is ahemistry  and, morhemistry . Trials hemistry .FLNA, UXT and the MAGE family genes,  [To date, most studies of CADs are based on immunohistochemical evaluation of AR expression in neoplastic cells. The AR gene is located on X chromosome at Xq11–12 , 7. In a MAGEC2) . Recent  MAGEC2) , 11. TheConsecutive cases were retrieved from the files of the Surgical Pathology Units of the Department of Biomedical and Neuromotor Sciences at Bellaria Hospital in Bologna , St. Vincent’s University Hospital in Dublin (Ireland) and Morgagni Hospital in Forlì .Cases were retained for the present study when they showed (a) ER and PR negativity and AR positivity; (b) the morphological and immunohistochemical profile consistent with the diagnosis of CAD according to recent guidelines ; (c) sufImmunohistochemistry was performed on an automated stainer  applying a pre-diluted monoclonal anti-androgen receptor  and anti-GCDFP-15  antibodies.Dual-colour FISH was carried out according to a standard protocol as previously described , 11, shoFISH for HER2 gene amplification status was routinely performed, at the time of diagnosis, applying specific probes  with the same method cited above.FISH analysis was carried out using an Olympus BX61 epifluorescence microscope  equipped with a 100-planar objective. For each case, a minimum of 60 non-overlapping nuclei was scored. Scoring was performed as previously described : total nHER2 results were scored following currently available guidelines .Methylation analysis was performed as previously described . BrieflyStatistical analyses were performed using commercially available software: QuickCalcs, which is an online tool for linear regression by GraphPad, and Excel, from Microsoft Corporation.P values smaller than 0.05 were considered to reflect a significant difference between groups.The relationship between FISH results and clinical and histopathological data was calculated using the generalized Fisher’s exact test that is part of the GraphPad QuickCalcs online tool. All investigations were conducted according to the principles expressed in the Declaration of Helsinki; the study was approved by the local Ethical Committee (Code: n. CE17133).Twenty tumours met the inclusion criteria and constituted the basis of the present study. Clinical and pathological features are summarized in Table n = 16) and grade 2 (n = 4) [All patients were female, aged 49 to 85 years (mean age 64 years). Tumours were graded as grade 3 n =  and gradpTNM  was as fIn 19/20 tumours, AR stained more than 95% of the neoplastic cells Fig. . In the HER2 was 3+ or amplified by FISH in 8 cases (40%).FISH analysis of the X chromosome was performed on all tumours included in the study and informative results were obtained in 13 tumours. In all cases, normal ducts served as an internal control. Tumours were eliminated from the study when the two signals (red and green) were not clearly evaluable.X chromosome copy number variation was observed in 13 of 13 tested tumours. Specifically, all except one (case 3) showed a proportion of neoplastic cells with loss of one X chromosome copy (monosomy) Fig. . The perIn three tumours , a neoplastic population with an additional X chromosome copy (polysomy) Fig.  was idenNot all additional copies of the X chromosome carried the AR gene leading to AR gene relative deletion. AR gene deletion was detected in 9/13 cases and affected from 2.43 to 56.17% (average 28.62%) of the neoplastic cells.Overall, AR gene loss (due to either X chromosome monosomy or AR gene deletion) was observed in all 13 cases tested. The neoplastic cell population showing loss of one AR gene copy varied from 5 to 84% (mean 48.93%). Minimal differences were observed between HER2-positive and HER2-negative cases  of the neoplastic cells.Non-neoplastic, non-apocrine cells present around the tumour showed AR monosomy in two cases, in 15.73% and 4.17% of the cell population respectively. The related tumours showed AR monosomy in 25.19% and 57.14% of the neoplastic cells (cases 4 and 14).AR, FLNA and UXT genes, and of the MAGE family genes , all present on the X chromosome, was evaluated. MAGEG1, a member of the MAGE family that maps to chromosome 15, served as a control as it is not affected by X chromosome inactivation in females. It was consistently non-methylated, as expected, as chromosome 15 is not subject to lyonization.All 20 tumours were tested, but informative DNA was obtained in 9 cases only due to DNA over-fixation problems. The methylation status of the In all tumours, the tested genes showed variation in methylation status, with respect to the methylation condition of a single chromosome (50% of methylation).The AR gene showed a mean methylation value in the CpG islands lower than 50% in all but one case case 20)   in spite of X chromosome monosomy and AR gene loss in a variable percentage of neoplastic cells. All cases showed variable levels of hypomethylation of the tested genes. Even if a statistically significant correlation was not detected, methylation levels were lower in cases with higher AR gene loss, thus suggesting that the residual AR gene was transcriptionally active.AR is a targetable molecule, widely used in the treatment of prostatic cancer . ProstatIn the present series, almost all CADs tumours showed X chromosome aneusomy, mostly due to monosomy. AR monosomy was also present, even if at lower levels, in in situ carcinoma.AR gene and its regulators. Unfortunately, the correlation between FISH and methylation results was possible in 6 cases only. In these cases, it was shown that AR gene monosomy was related to the hypomethylation status of the gene and most of its regulators.All tumours showed AR protein expression on immunohistochemistry, with greater than 95% of the neoplastic population staining positively in 19/20 cases. The AR protein expression can be explained by the hypomethylation status of the These results lead to two considerations: X chromosome aneusomy can play a role in the neoplastic transformation of mammary epithelium and may regulate therapeutic response to molecules for anti-AR therapy.AR gene monosomy. Furthermore, in 2 cases, AR monosomy was observed in the normal ductal cells surrounding the invasive carcinoma. X chromosome aneusomy has rarely been studied in breast carcinogenesis. Persons et al. [AR gene copy number aberrations have been demonstrated in male breast cancer. However, male breast cancer, in contrast with the female CADs here studied, showed mainly X chromosome polysomy and related AR gene copy number gain [The hypothesis of AR aneusomy role in the neoplastic transformation is supported by the fact that in situ duct carcinoma present in the FISH sections demonstrated s et al.  studied ber gain , 11.The second consideration focuses on the possible role of AR as a therapeutic target. Student et al.  reviewedAR gene status paralleled by a great variability of AR and its regulator methylation status. In most of the studied tumours, the AR gene was monosomic and its regulators showed a variable methylation status, with a prevalence of hypomethylation. These conditions lead us to hypothesize that the monosomic AR gene is transcriptionally active, explaining why most of the neoplastic cells show AR positivity on immunohistochemistry. Nevertheless, differences in AR gene status could lead to quantitative differences in AR protein production. It is plausible to hypothesize that AR protein can be reduced in AR monosomic neoplastic cells.All the studies published to date focus on AR expression evaluated by immunohistochemistry. The data here shown demonstrate a great variability of AR gene polysomy may lead to castration therapy resistance [In patients with prostatic cancer, it has been demonstrated that sistance , 19. TheThe present work has some limitations, including the low case number, inability to perform quantitative evaluation of AR protein with western blot analysis due to the lack of freshly fixed tissue and some compromised FISH and DNA studies due to fixation issues.AR transcriptional status may be quite variable. This variability may have an impact on response to AR deprivation therapy.Despite these limitations, the data here shown demonstrate that in breast CADs even if AR immunohistochemical expression is high, ESM 1(DOCX 18 kb)


Abiraterone, a novel androgen synthesis inhibitor, has been approved for castration-resistant prostate cancer (CRPC) treatment. However, most patients eventually acquire resistance to this agent, and the underlying mechanisms related to this resistance remain largely unelucidated. Lysine acetyltransferase 2 A (KAT2A) has been reported to enhance transcriptional activity for certain histone or non-histone proteins through the acetylation and post-translational modification of the androgen receptor (AR). Therefore, we hypothesised that KAT2A might play a critical role in the resistance of prostate tumours to hormonal treatment. In this study, we found that KAT2A expression was increased in abiraterone-resistant prostate cancer C4-2 cells (C4-2-AbiR). Consistently, elevated expression of KAT2A was observed in patients with prostate cancer exhibiting high-grade disease or biochemical recurrence following radical prostatectomy, as well as in those with poor clinical survival outcomes. Moreover, KAT2A knockdown partially re-sensitised C4-2-AbiR cells to abiraterone, whereas KAT2A overexpression promoted abiraterone resistance in parental C4-2 cells. Consistent with this finding, KAT2A knockdown rescued abiraterone sensitivity and inhibited the proliferation of C4-2-AbiR cells in a mouse model. Mechanistically, KAT2A directly acetylated the hinge region of the AR, and induced AR translocation from the cytoplasm to the nucleus, resulting in increased transcriptional activity of the AR-targeted gene prostate specific antigen (PSA) leading to resistance to the inhibitory effect of abiraterone on proliferation. Taken together, our findings demonstrate a substantial role for KAT2A in the regulation of post-translational modifications in AR affecting CRPC development, suggesting that targeting KAT2A might be a potential strategy for CRPC treatment. Prostate cancer (PC) is the second most commonly diagnosed malignancy in men worldwide , 2. SincIntra-tumour and extra-gonadal androgens are responsible for persistent androgen receptor (AR)-mediated growth in CRPC and are thus considered potential therapeutic targets . RecentlThe PTM of AR plays an important role in the resistance to hormonal treatment and the development of CRPC, including phosphorylation, acetylation, methylation, ubiquitination, and ubiquitin-like modifier (SUMO)-ylation, among others , 16. AceLysine acetyltransferase 2 A (KAT2A), a member of the HAT family, is located on chromosome 17q21.2 and is associated with enhancement of transcriptional activity through histone acetylation –29, histTo evaluate the role of KAT2A in the resistance of PC to abiraterone, in this study, we established a stable C4-2 cell line that was resistant to abiraterone acetate (C4-2-AbiR); this cell line is distinct from the C4-2B cell line, which is resistant to enzalutamide and also showed cross-resistance to abiraterone , 37. We 2 and 95% humidified air.A total of 87 sets of specimens of PC tissues and their adjacent normal prostate tissues were obtained from patients who underwent radical prostatectomy for prostate carcinoma at the Department of Urology of Union Hospital affiliated of Tongji Medical College between 2015 and 2019. The intraepithelial neoplasia samples would be excluded from tumour samples. We have acquired the approval from the Institutional Review Board of Tongji Medical College of Huazhong University of Science and Technology before we collected the samples. Consent was obtained for experimentation with human subjects. All specimens were classified according to the 2004 World Health Organization Consensus Classification and Staging System for prostate neoplasms. C4-2 and LNCaP were provided from Prof. Xiaoping Zhang and Prof. Jun Zhao . PC-3 and 293 T were obtained from Shanghai Cell Bank, Chinese Academy of Sciences . Cells were maintained in RPMI 1640 medium  with 10% foetal bovine serum  that contained 1% penicillin/streptomycin  at 37 °C in 5% CO2 and 95% humidified air. After 6 months of induction, the stable abiraterone-resistant cell line was obtained and named C4-2-AbiR.The starting treatment concentration of abiraterone acetate  in the parental C4-2 cell culture medium was 0.5 μM. At this concentration, the cells were stably passaged three times. The drug concentration was then increased, and the culture was continued. The concentration gradients of abiraterone acetate were 1 μM, 2 μM, 4 μM, 8 μM, 10 μM, 14 μM, 16 μM, 20 μM, 22 μM and 25 μM. Cells were maintained in phenol red-free RPMI 1640 medium  with 10% charcoal stripped fetal bovine serum  that contained 1% penicillin/streptomycin  at 37 °C in 5% COCell cycle detection were performed by flow cytometer . Cells were harvested by centrifuging at 1500 rpm for 5 min, fixed with ethanol at 4 °C overnight, and stained with PI. The percentage of cells in each cell cycle phase was analyzed.2 for 2-3 weeks, and the colonies with more than 50 cells were scored. The cell colonies were immobilized with 4% paraformaldehyde and dyed by crystal violet. When the counting of plates has been finished, we used formulation to calculate the rate of colony formation. (Rate = the number of colonies/ the number of seeded cells).C4-2-AbiR, C4-2 and LNCaP cells transfected with the plasmids were cultured in 6-wells plates at the density of 800–1000 cells per well. Plates were incubated at 37 °C in 5% COcDNA was synthesized from total RNA by using the iScript cDNA synthesis kit  following manufacturer protocols. Quantitative real-time PCR was performed by using the ABI Power SYBR Green PCR Master Mix  with the 7900 HT Sequence Detection System (Applied Biosystems). Glyceraldehyde 3-phosphate dehydrogenase was used as internal control. PCR primer pairs were synthesized by Sangon Biotech , and primer sequences are presented in Supplementary Table Total protein was isolated by using a RIPA lysis buffer (Beyotime Institute of Biotechnology), separated on SDS-PAGE gels, and transferred to PVDF membranes . Membranes were blocked by 5% nonfat milk in Tris-buffered saline with Tween-20, then incubated with primary Abs overnight at 4 °C and with corresponding secondary Abs . The following primary Abs were used: rabbit anti-human KAT2A , rabbit anti-human AR , rabbit anti-human PSA , rabbit anti-human acetylated lysine , rabbit anti-human His tag , rabbit anti-human Flag tag . Mouse anti-human β-actin  and rabbit anti-human lamin B1  were used as loading controls. Protein bands were visualized with ECL (Beyotime Institute of Biotechnology).2O2, followed by incubation with normal goat serum . After incubation with the primary antibody overnight at 4 °C, sections were washed with PBS and incubated with the biotinylated secondary antibody (Vector Laboratories), followed by incubation with Vectastain ABC Reagent (Vector Laboratories). The visualization signals were developed using diaminobenzidine , and the slides were counterstained with hematoxylin. The images were captured using an Olympus BX60 microscope . Immunoreactivity was scored based on a combination of both the percentage and intensity of positively stained tumour cells to generate an H-score. Staining intensity was divided into four categories as follows: no staining, 0; weak staining, 1; moderate staining, 2; strong staining, 3. H-score was determined according to the formula: (% of weak staining×1) + (% of moderate staining×2) + (% of strong staining×3), yielding a range from 0 to 300. Samples from patients were stained with KAT2A and AR and those from xenograft tumours were stained with KAT2A, AR, PSA and Ki67.Samples included those from 87 patients with PC and xenograft tumours in vivo. Samples were paraffinized, rehydrated, and blocked with 3% of HControl group and treatment group cells were plated on 96-well plates at 5000 cells/well and cultured with complete medium overnight. After 24 h, 10 ml of Cell Counting Kit- 8 solution  was added to each well and the plate was incubated for 2 h at 37 °C. This process was repeated for four days. Absorbance at 450 nm was measured on a microplate reader .We constructed seven types of plasmids which are plasmids overexpressing KAT2A, plasmids with knocked-down KAT2A, plasmids overexpressing AR, AR-K630A mutant plasmids, AR-K632A mutant plasmids, AR-K633A plasmids, AR-K630Q mutant plasmids . Plasmids and vectors were transfected into C4-2 and LNCaP with Lipofectamine 2000 according to protocol. C4-2 and LNCaP were infected with lentivirus overexpressing KAT2A .Cells were fixed in 4% paraformaldehyde, permeated by 0.3% Triton X-100, and blocked with 3% BSA for 1 h at 37°C, followed by incubation with primary KAT2A and AR antibodies. All data were analyzed via Nikon A1Si Laser Scanning Confocal Microscope .2 dishes were harvested, collected in lysis buffer, left on ice for 30 min, sonicated, and centrifuged at 15000 rpm for 15 min at 4 °C. Supernatants were collected. Each immunoprecipitation (IP) was carried out using 5-μg antibody and 500-μg protein. The precipitated proteins were collected using protein A + G beads, washed, eluted in boiling Laemmli sample buffer, and subjected to Western blotting. Briefly, 100-μg protein from each group was fractionated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes . The membranes were then blotted with primary antibodies, followed by the secondary antibody and developed with enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology). The primary antibodies used in this study were obtained from  and .Subconfluent proliferating cells in 100-cm4cells per well) 24 h before transfection. The pGL3-basic plasmids containing promoter of PSA and renilla luciferase reporter vectors (pRL-TK) were co-transfected with AR and KAT2A overexpression plasmids, to determine the combination level of AR and promoter of PSA. Besides, plasmids of the transcription binding sites of KAT2A (JASPAR database) were co-transfected with AR overexpression plasmids to determine the specific binding sites of KAT2A and AR. 48 hours after transfection, the firefly and renilla luciferase activities were measured with Dual-Luciferase® Reporter Assay System  as described in specification.PC-3 cells were seeded in 24-well plate (6 × 106 C4-2-AbiR cells with knockdown of KAT2A or control cells suspended in 100 μl of serum-free medium were injected subcutaneously into the armpit on the right side of the mice. Tumour volumes (V) were measured every five days based on measurements of length (L) and width (W) and calculated as V = (L×W2)/2. When the tumours reached 100–200 mm3, the mice were randomized equally, treated with vehicle or abiraterone acetate (60 mg/kg) and prednisolone (0.1 mg/kg) twice a week, and sacrificed ~40 days later (n = 5 each group). Mice that died before completion of experimental protocol were excluded from further data analysis. Investigators who performed endpoint analyses were blinded to group allocation.Three- to four-week-old castrated male nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Their care was in accordance with institution guidelines. Approximately 5×10Data of GDS2545 in Gene Expression Omnibus (GEO) database were used . Data from J B Welsh et al in Oncomine database were used . Data ofp values, which were considered significant at less than 0.05. Test of normal distribution would be performed to decide either parametric test or nonparametric test to be used. F test would be performed to compare variances between the groups to decide whether Welch’s correction should be used.All statistical analyses were performed using Prism 8.0 (GraphPad) and SPSS 22.0 (IBM Corporation). All in vitro experiments were repeated three times. All data are presented as the mean ± SD. Survival information was verified by Kaplan–Meier analysis and compared using the log-rank test. A two-tailed unpaired Student’s t test was used to determine the p < 0.05), disease-free survival (p < 0.0001), and PFS (p < 0.001) than those with low KAT2A expression  and C4-2-AbiR cell lines are shown in Fig. The ICTo explore the function of KAT2A in PC abiraterone resistance, we knocked it down in C4-2-AbiR cells using KAT2A-specific shRNA Fig. . AlthougAR is a key transcription factor that plays a vital role during PC progression, as well as in endocrine therapy resistance. Considering that high KAT2A expression was associated with abiraterone resistance, to explore the underlying mechanism, we next evaluated whether KAT2A induced resistance through the AR signalling pathway. First, we knocked down KAT2A expression and examined protein and mRNA levels of AR and the AR-targeted gene PSA in C4-2-AbiR cells. As shown in Fig. To further evaluate whether the AR signalling pathway is involved in the KAT2A-mediated resistance to abiraterone, we investigated the transcriptional function of AR by designing dual-luciferase reporter plasmids containing wild-type and mutant PSA promoters Fig.  and co-tKAT2A can act as an acetyltransferase for both histone and non-histone protein substrates, 31, 32.Mutation of the K630, K632, and K633 residues in the hinge region to alanine has been reported to cause a loss of AR acetylation . To confK630A and the acetylation-mimicking ARK630Q to determine the role of K630 in KAT2A-mediated AR translocation. K630Q mimicked the effect of KAT2A overexpression in PC-3 cells, whereas K630A could offset the effect of KAT2A overexpression  and prednisolone (0.1 mg/kg) twice per week. As shown in Fig. HATs, also known as lysine acetyltransferases (KATs), are a group of enzymes that can directly acetylate histone lysine residues to modify transcriptional function by weakening the interaction between histones and DNA and recruit DNA-binding proteins, which serve as docking sites , 45. SevIn hormone-sensitive PC, the cancer cells rely on androgen binding to the AR to induce tumourigenesis . Even afActivation of the AR signalling pathway is significant for the progression of CRPC . TransloStudies have shown that the nuclear translocation of AR is mediated by a bipartite nuclear localisation signal located within the hinge region, suggesting that the hinge region of AR has a strong correlation with its translocation into the nucleus , 54. ThrIn summary, the present data showed that KAT2A promotes resistance to abiraterone and that targeting KAT2A might reverse abiraterone therapy resistance in CRPC. The combination of targeting KAT2A with abiraterone treatment might provide a new therapeutic strategy for CRPC.Supplementary information


These proteins play roles in cell migration and adhesion but are also important in disease etiology, including in cancer angiogenesis and metastasis. While some structures of the soluble domains of these receptors have been determined, the conformations of the full-length receptor complexes are just beginning to be elucidated, especially within the context of the plasma membrane. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy allows direct insight into the formation of protein–protein interactions in the membranes of live cells. Here, we investigated the homodimerization of neuropilin-1 (Nrp1), plexin A2, plexin A4, and plexin D1 using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy. Consistent with previous studies, we found that Nrp1, plexin A2, and plexin A4 are present as dimers in the absence of exogenous ligand. Plexin D1, on the other hand, was monomeric under similar conditions, which had not been previously reported. We also found that plexin A2 and A4 assemble into a heteromeric complex. Stimulation with semaphorin 3A or semaphorin 3C neither disrupts nor enhances the dimerization of the receptors when expressed alone, suggesting that activation involves a conformational change rather than a shift in the monomer–dimer equilibrium. However, upon stimulation with semaphorin 3C, plexin D1 and Nrp1 form a heteromeric complex. This analysis of interactions provides a complementary approach to the existing structural and biochemical data that will aid in the development of new therapeutic strategies to target these receptors in cancer.Signaling of semaphorin ligands  The semaphorins are a large family of secreted and transmembrane ligands that regulate cell morphology and motility during development in a broad range of tissues . About 2Of the class 3 semaphorins, semaphorin 3A is the most well studied for its function as a chemorepellent in axon guidance and growth cone collapse, where deletion can lead to excessive axonal branching , 13, 14.Signaling is initiated when semaphorins bind to plexin receptors. Semaphorin 3C regulates downstream signaling in the presence of Nrp1 , plexin et al.  was unaffected by semaphorin 3A or semaphorin 3C stimulation. A complex between plexin D1 and Nrp1 was observed following incubation with semaphorin 3C; however, no interactions were observed following semaphorin 3A stimulation. The results presented here expand upon previous interaction studies by including multiple receptor and ligand pairs to begin resolving the full interaction profile for this important protein family. Advances in understanding this local network of protein interactions will aid in the development of new therapeutic strategies that target these receptors.2  fusion in each single cell measurement, which varied between 85 and 1245 molecules/μm2  and mCherry (mCh) fusion constructs to determine their degree of homodimerization. PIE-FCCS data were collected from single live Cos-7 cells expressing the tagged protein of interest at surface densities ranging from 85 to 1245 molecules/μm2 . From ea2 . The Nrp1 data had a median of 0.14, which is consistent with strong dimerization; however, the wide distribution of fc values over 0.20 suggests that Nrp1 can also form small homotypic multimers as reported previously  indicating a weaker dimer affinity compared with plexin A4. To the best of our knowledge, there has been no investigation of the oligomerization state of plexin D1, except for a computational prediction that the isolated transmembrane helix is expected to dimerize to a similar extent as other plexin transmembrane domains (fc = 0.01) suggests that full-length plexin D1 does not dimerize in the given concentration range. The diffusion coefficients for each receptor support the interpretations of the fc values (B), with higher mobility observed for monomeric plexin D1 compared with plexin A2 and plexin A4. Nrp1 has an average diffusion coefficient of 0.26 μm2/s, consistent with the formation of multimers as this is significantly slower than dimeric plexin A2 and plexin A4, where the average diffusion coefficients are 0.39 and 0.37 μm2/s, respectively. Plexin D1 has the fastest average diffusion coefficient, 0.59 μm2/s, adding to the evidence that it is monomeric.The receptor A. The Nreviously , 37. ThePIE-FCCS . The ple domains . The neac values B, with hi.e., plexins, VEGF receptor, mesenchymal–epithelial transition factor). However, conflicting reports exist regarding the interactions of these receptors in heteromeric complexes before and after stimulation. Man et al. . This lack of cross-correlation indicates that unstimulated receptors have negligible propensity to dimerize with Nrp1 in the live cell plasma membrane. The average diffusion coefficient of Nrp1 expressed with plexin D1 showed a modest increase from 0.26 to 0.34 μm2/s compared with when it was expressed without plexin D1 (B). This suggests that Nrp1 may form oligomers when expressed alone but shifts toward dimerization when in the presence of coreceptors as previously reported . With Nrp1 acting as a coreceptor for many other receptors , the lack of cross-correlation in our assay may also be due to a competition between plexins and other endogenous receptors, that is, the plexin binding to Nrp1 is too weak to compete off these interactions.Nrp1 is involved in class 3 semaphorin signaling as well as other ligands like vascular endothelial growth factor (VEGF) but cannot transduce the signal without expression of additional receptors . This indicates that neither class A plexin forms a complex with plexin D1 under nonstimulatory conditions. However, the median fc value for plexin A2 and plexin A4 is 0.08, suggesting the presence of heterodimers in live cells. The average diffusion coefficient of both class A plexins is unchanged when expressed alone or with the other class A plexin (B). This allows us to conclude that the complex formed is most likely a heterodimer and not a larger multimer. This is the first observation, to the best of our knowledge, that class A plexins can be involved in heterodimers with other class A plexins prior to ligand stimulation.To determine the degree of interaction between each plexin receptor pair, we conducted pairwise coexpression of each receptor combination in Cos-7 cells and collected PIE-FCCS data . The reason for the dramatic increase in cross-correlation is likely because of the reduced competition with the plexin A4 homodimers and possibly the release from an autoinhibited structure (see B). The plexin A4 homodimer is now abrogated, reducing competition with the heterodimer, and causing the dramatic increase in codiffusion. These experiments show the necessity for heterodimerization analysis by PIE-FCCS to begin building a model that incorporates the full complexity of membrane protein interaction networks.Based on a crystal structure from Kong et al. , class Aet al. . We coex  and semaphorin 3C (0.59–0.68 μm2/s), respectively  in the live cell plasma membrane. Following semaphorin 3C stimulation, plexin D1–Nrp1 did show substantial increase in heterodimerization (fc = 0.13). These changes in oligomerization state of plexin D1 and Nrp1 were supported by changes in the effective diffusion coefficient (B and C). The average diffusion coefficient of plexin D1 decreased by 20% (0.62–0.50 μm2/s), indicating increased molecular weight and a shift from monomer to heteromeric complex (B). The Nrp1 diffusion coefficient was significantly higher than in the homodimer experiments (0.31 compared with 0.26 μm2/s) but does not significantly decrease upon stimulation with semaphorin 3C (0.34–0.31 μm2/s). This result is consistent with a shift from Nrp1 homomultimers to heteromeric complexes (C). fc distribution for each combination of plexin receptors was relatively unchanged following semaphorin 3C stimulation.We next tested the interaction between Nrp1 and each plexin receptor in the presence of semaphorin ligands. Each receptor combination was coexpressed in Cos-7 cells, and PIE-FCCS data were collected as in the previous experiments. Using the same concentration and incubation conditions from the previous section, the coexpressed receptors were stimulated with recombinant semaphorin 3C. The n et al.  and Toyo . Average diffusion coefficients for plexin D1 and Nrp1 were unchanged compared with homotypic and heterotypic interaction rates; however, plexin A4 and plexin A2 diffusion coefficients decreased (C). Both plexin A2-eGFP and plexin A4-eGFP have their lowest average diffusion coefficient when expressed with Nrp1 and stimulated with semaphorin 3A compared with expression of the plexin alone, 0.39 to 0.30 μm2/s and 0.37 to 0.28 μm2/s, respectively. Nrp1-mCh diffusion was not significantly changed under any condition. Overall, these data do not give any clear indication of a transition to heterodimer after semaphorin 3A binding as was seen for plexin D1 and Nrp1 following semaphorin 3C binding. However, some caution is advised when interpreting these negative results. PIE-FCCS measurements of heterodimers are affected by the stability and dynamics of the heterodimer as well as any competition with homodimers and heterotypic interactions with other endogenous receptors. Until the full network of membrane protein interactions can be resolved, it is difficult to rule out low-affinity interactions based on negative PIE-FCCS studies. Alternatively, it is also possible that three receptors may be necessary to form a stable signaling complex and that pairwise expression of exogenous receptors is insufficient to drive the formation of the full signaling complex. This type of heteromeric complex has been suggested in previous studies but not observed directly in live cell biophysical assays  inhibits the homodimerization as we previously reported . When plfc value falls within the range expected for simple dimerization . There are several possible explanations for these results. First, the timescale of the association could be short. Our single cell measurements were performed during a period of 10 to 70 min after ligand addition. It could be that the formation of the complex is transient and thus appears weak in the average cross-correlation measurements. A second reason could be that there are multiple competing interactions with endogenous proteins. Because of the relative affinities of these competing interaction partners, there may be an ideal set of expression levels under which the heteromeric complex reported in the co-IP and crystallography studies is visible via PIE-FCCS. As stated previously, PIE-FCCS measurements of heterodimerization are affected by the stability and dynamics of the heterodimer as well as any competition with homodimers and heterotypic interactions with other endogenous receptors. Until the full network of membrane protein interactions can be resolved, it is difficult to rule out low-affinity interactions based on these negative PIE-FCCS results.While previous reports have suggested that Nrp1 and class A plexins form a complex after stimulation with semaphorin 3A, our analysis by PIE-FCCS does not provide strong supporting evidence. A large increase in the ficients , B and Cper se. Studies of plexin B1 have led to a model in which pre-existing dimers are not just conformationally altered upon ligand binding, but that plexin–semaphorin 2:2 heterodimers may also associate to form larger order complexes . We were not able to identify a report of the in vivo density of plexin receptors, but the expression is low in our model Cos-7 cells as seen in in vivo will be dependent on cell type, expression level, and dimerization affinity. Future work will need to account for these parameters.One important consideration of any quantitative live cell biophysical method is the optimal concentration range of the experiment. For single-molecule imaging, this range is less than one molecule/μmles/cell  or betweOverall, the work here has investigated only a subset of the potential interactions within the plexin–neuropilin–semaphorin protein family. More receptors and ligand combinations will need to be analyzed to establish a more expansive and holistic understanding of how membrane protein–protein interactions regulate plexin–semaphorin signaling. Because of the large number of ligands and receptors , this is a time- and resource-intensive undertaking. The work we presented here lays the groundwork for such a comprehensive study. PIE-FCCS is an ideal method for quantifying these interactions in a live cell environment. Combined with cell signaling and high-resolution structure studies, it will be possible to resolve the function role of receptor homodimerization and heterodimerization in this important signaling axis. This work will also reveal how dysregulated signaling by plexins and neuropilins influence disease states, which will enable new approaches for designing therapeutic strategies.Each of the full-length human receptor proteins were cloned into pEGFP-N1 and pmCh-N1 vectors for mammalian expression. Cloning of plexin A2-eGFP (accession no.: O75051) and plexin A2-mCh was carried out by inserting the plexin A2 sequence into EcoR1 and Kpn1 sites of the vectors. The cloning primers areForward: 5′ ACTGAATTCATGGAACAGAGGCGGCCCTGGCCCC 3′ and Reverse: 5′ ACTGGTACCGTGCTCTCAATGGACATGGCAT TAATGAGCTG 3′. Plexin D1-eGFP (accession no.: Q9Y4DY) and plexin D1-mCh were cloned by using EcoR1 and BamH1 sites in vectors and the plexin D1 internal Sac1 to amplify two pieces followed by a three-way ligation. The cloning primers areForward 1: 5′ AATGAATTCATGGCTCCTCGCGCCGCGGGCGGCGCACCCCTTAGCGCCCGGGCCGCCGCCGCCAGCCCCCCGCCGTTCCAGACGCCGCCGCGGTGCCCGGTGCCGCTGCTGTTGCTGCT 3′;Reverse 1: 5′ GCACCAGGACCTGGAGCTCGGAGCCTACATGG 3′;Forward 2: 5′ CCATGTAGGCTCCGAGCTCCAGGTCCTGGTGC 3′;Reverse 2: 5′ AATGGATCCCGGGCCTCACTGTAGCACTCGTAGATGTTGTCCTCCATCAAAGCCAC 3′.Cloning of Nrp1-eGFP (accession no.: O14786), Nrp1-mCh, plexin A4-eGFP (accession no.: Q9HCM2), plexin A4-mCh, plexin A4ΔSema-eGFP, and plexin A4ΔSema-mCh was performed as previously described . The pleCos-7 cells were cultured and transiently transfected using standard procedures . BrieflyPIE-FCCS data collection was performed as previously described , 70. BriF(t) was compared with the intensity at a later time F(t + τ) and the self-similarity as a function of the later time allowed for interpretation of quantitative information such as diffusion and the number of particles. Intensity fluctuations were separated into 10 μs bins and subjected to the correlation algorithm in Equation For each single-cell measurement, five acquisitions of 10 s were recorded at the peripheral membrane area. Each intensity fluctuation was subjected to PIE gating before the autocorrelation and cross-correlation analysis. In an autocorrelation analysis, the intensity at time The five acquisitions from each single-cell measurement were averaged together to remove perturbations such as cell movement or bright clusters. Once individual curves from each cell are averaged, a least squares fitting to a 2D diffusion model is used, and includes fitting parameters for the triplet state Equation .(3)G(τ)=With autocorrelation, it is possible to infer protein diffusion by using the timing of intensity fluctuations. The half value decay time  of zero for a noninteracting species and a fraction correlated of one for an interacting species. However, we must take certain considerations into account when interpreting live cell fluctuation results. Our laboratory's previous publications established a set of control constructs with a myristoylation anchor fused to dimerization motifs and an FP allowing for interpretation of fc values for monomers, dimers, and higher order oligomers ., 67.fc) Samples for Nrp1, plexin A2, plexin A4, and plexin D1 endogenous expression in Cos-7 cells were collected 24 h after passaging. Samples for transient expression of Nrp1-eGFP, plexin A2-eGFP, plexin A4-eGFP, and plexin D1-eGFP in Cos-7 cells were transfected with 2.5 μg plasmid DNA 24 h after passaging and collected 24 h posttransfection. Cells were lysed using radioimmunoprecipitation assay lysis buffer supplemented with protease inhibitors . Western blotting experiments with these samples were carried out to confirm the expression of the samples. Primary antibodies used are Nrp1 , plexin A2 , plexin D1 , and plexin A4 .All data that support the findings of this study are contained within the article and its supporting information.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Long noncoding RNAs (lncRNAs) are involved in a variety of human immune diseases. However, the expression profile and precise function of lncRNAs in allergic rhinitis (AR) remain unknown. In the present study, genome-wide analysis of lncRNA expression was performed in the nasal mucosa tissue and mRNA regulatory relationship was examined among patients with or without AR.Microarray assays were performed and the differential expressions of lncRNAs or mRNA were verified through RT-PCR. The lncRNA functions were annotated using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The potential regulatory relationships between lncRNAs and the co-expressed mRNAs were analyzed using Cytoscape software. The expressions of specific lncRNAs and mRNAs were examined using an in vitro cell model.P < 0.05). GO and pathway analysis indicated that the lncRNA–co-expressed mRNAs were enriched in several biological processes and cellular signaling pathways related to AR, such as positive regulation of the integrin biosynthetic process, cell adhesion, and leukocyte transendothelial migration. Some lncRNAs regulated the co-expressed genes in a cis- and/or trans-regulatory manner. Furthermore, allergen exposure significantly increased the expression of lnc-CXCL12-4, CXCL12, and CXCR4 in BEAS-2B cells compared to untreated cells (P < 0.01).A total of 57 lncRNAs and 127 mRNAs were dysregulated in the nasal mucosa tissue of patients with AR, compared to those of patients without AR (fold change > 2.0 and The results of the present study suggest that lncRNAs participate in the biological pathways related to AR. Leukocyte transepithelial migration may be a potential target for lncRNAs to regulate allergic inflammation and CXCL12/CXCR4 axis plays an important role in the inflammatory process of AR.The online version contains supplementary material available at 10.1186/s12920-021-00949-4. Allergic rhinitis (AR) is an IgE-mediated upper airway inflammatory response, which is becoming a global health problem . AR is cIt is well known that most of the genome is transcribed into RNA, but only a very small percentage of the transcripts are protein-coding genes, accounting for only 1.5–2% . TherefoThe aim of the present study is to examine the expression profiles of lncRNAs and mRNAs in AR. We identified the differential expression of lncRNAs and mRNAs in nasal tissues from AR and non-AR patients using microarray assays. Moreover, we analyzed the potential functions of the differentially expressed lncRNAs via bioinformatics analysis and validated the meaningfully enriched pathway using an in vitro cell culture model.A total of 8 AR patients and 10 non-AR patients were admitted to the Department of Otolaryngology-Head and Neck Surgery, Shanghai East Hospital between 2016 and 2019. All of the patients with AR had a positive skin prick test reaction only to dust mites and were diagnosed based on their medical history, nasal endoscopic examination, and allergen skin prick test. None of the participants had received topical or systemic glucocorticoid therapy for 4 weeks before tissue collection. Nasal mucosal tissues were obtained surgically from the inferior turbinates of the patients. The harvested samples were snap-frozen in liquid nitrogen and stored at –80℃. All patients had nasal septum deviation and were scheduled to undergo septoplasty and partial removal of the inferior turbinates. The study conforms to the standards of the Declaration of Helsinki. Patients who had a history of previous nasal surgery, smoking, autoimmune diseases, concurrent sinusitis, or systemic diseases were excluded from our study. Patient clinical characteristics are summarized in Additional file For the lncRNA and mRNA microarrays, total RNA was extracted from 100 mg of nasal mucosal tissue from 3 AR patients and 3 non-AR patients using TRIzol reagent . Total RNA was quantified by a NanoDrop ND-2000 spectrophotometer  and the RNA integrity was assessed using an Agilent 2100 Bioanalyzer .Total RNA labeling, microarray hybridization, and washing were performed using an Affymetrix Human OE lncRNA array  based on the manufacturer’s instructions. The microarray profiling was conducted in the laboratory of Shanghai OEBiotech . This microarray contains probes for 25,986 mRNAs and 66,741 lncRNAs.P value < 0.05 (Student’s t-test). The unsupervised hierarchical clustering of differentially expressed lncRNAs and mRNAs was carried out. The differentially expressed mRNAs were input into the DAVID database (http://david.abcc.ncifcrf.gov) for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation classification.Raw data were extracted using the Affymetrix GeneChip Command Console . RMA (Robust Multichip Average) normalization for both gene and exon level analysis was performed using Expression Console . GeneSpring software  was employed to complete subsequent data processing. After log2 transformation of the raw signals, differential expression of lncRNAs and mRNAs was defined by the absolute value of fold change (> 2.0) and −ΔΔct method.Total RNA was extracted from the nasal mucosal tissue from 8 AR and 10 non-AR patients using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The first strand cDNA was reverse-transcribed from 500 ng of total RNA using PrimeScript™ RT Master Mix . SYBR Premix Ex Taq™  was used to conduct real-time PCR using an ABI 7500 Real-Time PCR System . The specific primer sequences used in qRT–PCR are shown in Table 2 at 37℃. All experiments were performed with BEAS-2B cells at 80–90% confluency. Cells were exposed to different concentrations of ovalbumin (OVA) (100 μg/mL) or house dust mite (HDM)  (40 μg/mL) for 4 h.Human bronchial epithelial cell line BEAS-2B was purchased from ATCC  and cultured in BEGE medium with a Bullet Kit . All incubations were carried out in a humidified atmosphere of 5% COP values of PCC < 0.05 and absolute values of PCC > 0.8.Before predicting the possible functions of lncRNAs in AR, a correlation analysis of lncRNAs and mRNAs involved in allergic inflammation was carried out . AccordiP < 0.05 and false discovery rate (FDR) < 0.01. The most enriched annotations reflected the potential functions of the co-expressed lncRNAs.For function prediction of lncRNAs, an enrichment analysis of the co-expressed mRNAs was performed using the hypergeometric cumulative distribution function . The enrP < 0.05 and FDR < 0.01). Then we predicted that these lncRNAs possibly regulated the target genes in a trans-regulatory manner. The lncRNA–TF–mRNA network was constructed based on the interactions between the lncRNAs and the co-expressed target mRNAs. We selected the top 10 prediction regulating relationships with the highest prediction reliability to construct the core network map using Cytoscape software.To explore the potential target genes in AR, cis- and trans-regulatory analysis of the differentially expressed lncRNAs was performed. For cis-regulatory analysis, we identified the cis-regulated genes when the co-expressed mRNA loci were within 100 kbp upstream and downstream of the given lncRNA. Another regulatory mechanism of the specific lncRNAs in the expression of certain genes involves the factors mediating chromatin transcription (TFs) , 14. So t-tests. P < 0.05 was considered statistically significant.All data were expressed as mean ± standard deviation and analyzed using IBM SPSS Statistics, Version 22 . The differences in expression of lncRNAs and mRNAs in nasal mucosal tissue between AR and non-allergic patients were analyzed using Student's We profiled the expression patterns of lncRNAs and mRNAs associated with AR via microarrays. Our data showed that a total of 57 lncRNAs were differentially expressed in the nasal mucosa from AR and non-AR patients, with 22 upregulated and 35 downregulated, as indicated by the volcano plots and heat maps Fig. a, b. SimTo validate the microarray results, we randomly chose 4 lncRNAs and 2 mRNAs from the differentially expressed lncRNAs and mRNAs for qRT–PCR. As Fig. There are thousands of co-expression relationships between the differentially expressed lncRNAs and mRNAs. We analyzed the co-expression relationships of the top 500 pairs by PCC and constructed a co-expression network using Cytoscape software Fig. . The visThe potential functions of the lncRNAs were predicted by the GO and KEGG pathway annotations of their co-expressed mRNAs. The GO categories are biological process, molecular function, and cellular component. GO analysis of the co-expressed mRNAs revealed that the most enriched annotations were involved in positive regulation of the integrin biosynthetic process, cell adhesion, focal adhesion, inflammatory response, extracellular matrix, T cell receptor complex, cell junction, and intracellular calcium activated chloride channel activity. We counted and summarized the top 20 GO annotations with the most credentiality Fig. a–c. In aLncRNAs may regulate the nearby genes in a cis-regulatory manner. Therefore, we screened the chromosomal co-expressed mRNAs 100 kbp upstream and downstream of 57 differentially expressed lncRNAs and identified 35 lncRNAs with 41 potential cis-regulated mRNAs. The lncRNAs and the potential cis-regulated mRNAs are included in Table We calculated the significance of enrichment of each co-expressed mRNA in TFs from the Encyclopedia of DNA Elements, and identified 143 lncRNA–TF pairs, including 50 lncRNAs and 47 TFs. We selected the top 100 lncRNA–TF pairs with the most credentiality and created the lncRNA–TF two-element network relationship using Cytoscape software   Fig. b, c. The+ T cells from AR mice [LncRNAs are considered to be important regulators of cellular process, such as development, differentiation, and metabolism, through affecting gene expression and cell homeostasis , 16. Evi AR mice . A chang AR mice . These sIn the present study, we assessed genome-wide lncRNA expression patterns in the nasal mucosa from patients with and without AR by microarray analysis, and predicted their possible functions by analyzing the co-expressed mRNAs. Moreover, we also used an in vitro model mimicking the allergen exposure environment of airway epithelium to verify the predicted results. Our results indicated that 57 lncRNA and 127 mRNA transcripts were identified as being differentially expressed between the two groups, including 22 upregulated and 35 downregulated lncRNAs, and 43 upregulated and 84 downregulated mRNAs, respectively. The correlation between some of the differentially expressed mRNAs and AR has been reported, such as ANO1, THY1, CXCL12, and IL33. The expression of ANO1 is higher in AR patients than in healthy controls and hypersecretion of fluid and mucus in AR is closely related to ANO1 . THY1 geThe functions of the lncRNAs have not yet been fully annotated and the most common method for their functional prediction is through referring to the functional annotations of their co-expressed mRNAs . As showDue to the variety of functions of lncRNAs, their molecular regulatory mechanism remains unknown . PreviouBesides genetic and lifestyle-related factors, AR is also affected by the composition of inhaled air. The respiratory epithelial cells may mediate parts of the innate and adaptive immunity by their antigen presentation, phagocytosis, cytokine secretion, and pattern recognition abilities . The epiA series of aberrantly expressed lncRNAs may participate in the regulation of target protein-coding genes involved in the biological pathways related to AR in cis- and trans-regulatory manners. On the basis of these findings, we propose that the CXCL12/CXCR4 axis plays a very significant role in the inflammatory process of AR, which is regulated by lnc-CXCL12-4. Leukocyte transepithelial migration may be a potential target for lncRNAs to regulate allergic inflammation.Additional file 1: Table S1. Clinical characteristics of AR and non-AR patients.Additional file 2: Table S2. The differentially expressed mRNAs and lncRNAs.