Study On The Mechanism Of PRMT5 Cascade Regulation By LncRNA ZFAS1/miR-150 To Promote Invasion And Metastasis Of Prostate Cancer

Background and objective: Prostatic Cancer(PCa)is one of the most common urogenital tumors in men.According to the 2018 global cancer statistics,prostate cancer has the second highest incidence and the fifth highest mortality rate in men.It is estimated that there will be nearly 1.3 million new cases of prostate cancer and360000 deaths in 2018.In recent years,the incidence of prostate cancer in China has been increasing significantly.Although radical prostatectomy and radiation therapy remain the primary choice for localized stage of PCa,there is no effective treatment for patients with recurrences or metastatic disease,and patient who develop into castration-resistant prostate cancer(CRPC).Therefore,it is necessary to explore new and effective treatment methods.Protein arginine methyltransferases(PRMTs)play an important role in regulating the post-translational modification of various tumor-related proteins,including epigenetic histone modification and non-histone modification.A large number of studies have found that PRMTs family members are widely involved in regulating the development of a variety of tumors,and PRMT5 is the most deeply studied PRMTs family member.At present,the expression of PRMT5 in PCa,its regulatory mechanism and its correlation with PCa have not been studied.This study will systematically study the expression,function and regulatory mechanism of PRMT5 in PCa,and provide a theoretical basis for PRMT5 to become a new diagnostic,prognostic marker and therapeutic target of PCa patients.Methods: In the first part of our research,we downloaded the RNA-sequence datas in publically available data from the TCGA matched normal samples spanning33 cancer types and analyzed the expression of PRMTs family with the survival times of cancers.DAVID system was used to study the potential function of PRMTs in PCa,and its target genes were systematically and comprehensively annotated.Cytoscape software was used to demonstrate the regulatory relationship between PRMTs and target genes.Then,We constructed the co-expression gene network.With the comprehensive analysis of the PRMT5 expression of GEPIA database and GSE21032 database involved in PCa,Taylor database analysis confirmed the PRMT5 expression was a correlation with the the survival times of cancers.We Collected clinical tissue samples of PCa and evaluated the expression of PRMT5 and ki-67 by immunohistochemistry,and then detected the expression levels of PRMT5 and other proteins were by Western blot.Furthermore,we analysed the relationship between PRMT5 expression and clinicopathological characteristics of PCa,evaluated the expression level of PRMT5 and the prognosis of PCa with Kaplan-meier method and Cox proportional risk model and further verified RMT5 expression was in PCa cell lines.Next,we constructed the co-expression gene PPI network and detected 17 downstreamcore genes involved in PRMT5 by RT-PCR in down-regulated in PC-3and DU145 cell lines.In the second part of the study,we overexpressed or knocked down PRMT5 in PCa cell lines LNCa P,DU145 and PC-3 cells.After transfection efficiency was detected,the effect of PRMT5 on cell proliferation of PCa cells was detected by CCK-8 assay,the effect of PRMT5 on cell cycles of PCa cells were detected by Flow cytometry,the effect of PRMT5 on apoptosis of PCa cells were detected by Annexin V/PI,the effect of PRMT5 on migration and invasion of AR negative PCa cells were detected by transwell assay.After the expression of PRMT5 was knocked down in PC-3 and DU145 cells,Western blot was used to detect the expression of E-cadherin and Vimentin,the key genes in the EMT process.The stable transfection cell line with PRMT5 knocked down in LNCa P and PC-3 cells was constructed,and the xenograft tumor model of PCa in nude mice was established,and the growth curve of xenograft tumor in nude mice was plotted.HE staining was used to detect the morphology of PCa cell and lymph node metastasis of subcutaneous transplanted tumor.Immunohistochemistry and Western blot were used to detect the expression level of PRMT5 in transplanted tumors of nude mice.In the third part of the study,lncRNAs with significant correlation with PRMT5 expression level in 491 PCa samples were identified by analyzing TCGA database.PRMT5-lncRNAs with Pearson correlation coefficient of >0.4 were selected for further study.Using the DIANA-Lnc Base database,TARGETSCAN predicted PRMT5-targeted miRNAs.A ce RNA network was constructed to regulate PRMT5 in PCa.Then,using STARBASE 2.0 database,miRNAs interacting with PRMT5co-expressed lncRNAs were found.In order to increase the credibility of the results,we further screened lncRNAs with common MRE with PRMT5 by referring to database information such as mi Base,Target Scan,Pic Tar,Miranda.The ce RNA network was then rendered using Cytoscape software.The correlation between the expression levels of these lncRNAs and PRMT5 in PCa samples was further verified by using GEPIA database.PRMT5 was overexpressed in PC-3 cell lines,and the expression of these lncRNAs were detected by RT-q PCR.The expression of lncRNA ZFAS1 in PCa and its relationship with the survival time of PCa patients were analyzed by analyzing TCGA database.Dual fluorescence assay confirmed that PRMT5 and ZFAS1 were both target genes of miR-150.ZFAS1 was knocked down in PC-3 cells,and the effect of PRMT5 on migration and invasion of PC-3 cells was detected by transwell assay.The stable transfection cell line with ZFAS1 knocked down and PRMT5 overexpressed in LNCa P and PC-3 cells were constructed.and the xenograft tumor model of PCa in nude mice was established,and the growth curve of xenograft tumor in nude mice was plotted.HE staining was used to detect the morphology of PCa cell and lymph node metastasis of subcutaneous transplanted tumor.Immunohistochemistry and Western blot were used to detect the expression level of PRMT5 in transplanted tumors of nude mice.Results: In the first part,we analyzed the RNA-sequence datas in publically available data from the TCGA matched normal samples spanning 33 cancer types.We found that the PRMTs family were differential expression in a wide range of cancers.Unexpectedly,PRMT3,PRMT4/CARM1,PRMT5,PRMT6 and PRMT7 were uniformly highly expressed in prostatic cancer(PCa),suggesting that these genes play an important regulatory role in the occurrence and development of PCa.We explored an association between PRMTs expression and the survival times of cancers based on TCGA database,and found that high PRMTs expression associated with poor prognosis in many kinds of tumors.Then,We selected the top 200 key genes regulated by PRMTs(PRMT1-8)in GEPIA and constructed the co-expression gene network.We further validated PRMT5 was the heart of the PRMTs family.Therefore,in our present study,the underlying mechanisms of PRMTs in PCa were further explored and predicted by bioinformatics approaches,including screening target genes-related PRMTs.Automatic GO analysis of Molecule Annotation System(MAS3.0)was used to analyze the function of predict target genes.The result of bioinformatics prediction shows that PRMTs are widely involved in many biological processes such as RNA production,processing,splicing and transportation.More interestingly,we found that PRMTs were involved in chromatin remodeling,transcription,histone H3-K4 methylation and other biological processes,while our results are consistent with previous findings on up and reflect high accuracy prediction.Comprehensive analysis of the expression profiles of GEPIA database and GSE21032 database involved in PCa,we found that high PRMT5 expression was significantly associated with PCa,even in metastatic PCa.Which suggested that PRMT5 took play the role of proto-oncogene in PCa and participated in the metastasis regulation possiblly.Taylor database analysis showed that high PRMT5 expression was a significant positive correlation with the probabilities of distant metastasis-free survival(DMFS)and overall survival(OS).These results suggest that PRMT5 might be used as a diagnostic target for PCa.Furthermore,We confirmed that PRMT5 expression in PCa tissues is significantly higher than that in benign para-carcinoma tissues,and PRMT5 expression correlates positively with clinical stage.Expression of PRMT5 in PCa was significantly correlated with various clinicopathological characteristics,such as pathological T stage,clinical stage,distant metastasis and preoperative PSA level,ki-67 expression,and the state of surgical margin.The clinical datas showed that high expression of PRMT5 was positively correlated with shorter OS and DMFS in PCa patients.Further studies are necessary in order to conclude the association between PRMT5 expression and prognosis of PCa.We carried on a high throughput analysis of the level of PRMT5 expression and follow-up of 116 patients with PCa.Kaplan-Meier estimates and Cox proportional hazard models were used to evaluate PRMT5 expression and prognosis of PCa.Patients at risk are indicated high PRMT5 expression with poor OS and DMFS.Therefore,we will predict that PRMT5 is an independent poor prognostic factor for biochemical recurrence and overall survival time of patients with PCa.At the cellular level,PRMT5 m RNA expression was evaluated in PCa cell lines by q PCR,which was more than benign prostatic hyperplasia cell line RWPE-1,especially in PC-3 cells displayed the highest expression.Next,17 downstreamcore genes involved in PRMT5 were identified based on their levels of interaction via analyzing our PPI network.q PCR analyses revealed that following the knockdown of the PRMT5 gene,m RNA levels of the POLR1B、HSPA8、CCT4、RAN、NCBP1、RRP1、NSUN4、NLE1 genes were significantly downregulated.In the second part,we first explored the function and role of PRMT5 in PCa.When PRMT5 was highly expressed or knocked down in DU145,PC-3 and LNCa P cells,it was found that PRMT5 could promote the proliferation and cell cycle of AR positive PCa cells,inhibit the apoptosis of AR positive PCa cells,and there was no significant effect on the proliferation and cell cycle of AR negative PCa cells.However,PRMT5 can promote the metastasis and invasion of AR negative PCa cells.When we knocked down the expression of PRMT5,the expression level of PRMT5 in PC-3 and DU145 cells significantly decreased,the expression level of E-cadherin protein increased and the expression level of Vimentin protein decreased,the difference was statistically significant.The above experiments confirmed that PRMT5 promoted AR negative PCa cell metastasis and invasion by regulating EMT process.Subcutaneous PCa tumorigenesis in nude mice further verified that PRMT5 can affect the proliferation ability of AR positive PCa cells and the metastasis ability of AR negative PCa cells.In the third part,through the analysis of TCGA data,we found that the expression level of lncRNA lncRNA ZFAS1 in PCa samples was significantly higher than that in normal PCa samples.The progression-free survival time of PCa patients with high expression of lncRNA lncRNA ZFAS1 was significantly lower than that of PCa patients with low expression of lncRNA lncRNA ZFAS1.Cell function experiments showed that knockdown of lncRNA ZFAS1 significantly inhibited the proliferation and metastasis of PCa cells.Through the analysis of the public data,we found that the expression of lncRNA ZFAS1 and PRMT5 presented a significant positive correlation.In addition,the expression of miR-150 had significant negative correlation with lncRNA ZFAS1 and PRMT5 in PCa.Dual fluorescence assay confirmed that both PRMT5 and lncRNA ZFAS1 were target genes of miR-150.In vivo and in vitro rescue experiments showed that LncRNA ZFAS1 can be used as an ce RNA to absorb miR-150,regulate the expression of PRMT5 and promote the metastasis and invasion of PCa.Conclusions: TPRMT5 is highly expressed in public databases,PCa clinical tissue samples and PCa cells,and its high level of expression is significantly correlated with the clinicopathological characteristics and poor prognosis of PCa patients.PRMT5 regulates the metastasis and invasion of AR negative PCa cells by EMT process.LncRNA ZFAS1 can regulate PRMT5 expression by ce RNA adsorption of miR-150 and affect the process of proliferation and metastasis in PCa.The study of lncRNA ZFAS1-miR-150-PRMT5 axis is helpful to further understand the molecular mechanism of prostate tumor occurrence,development and metastasis.In addition,this study provides a new diagnostic,prognostic marker and therapeutic target for PCa patients.