MiR-30a Inhibits Androgen-independent Growth Of Prostate Cancer Via Targeting MYBL2,FOXD1 And SOX4

Prostate cancer(PCa) is one of the most common malignant tumors in men,and its morbidity and mortality are in the forefront of male malignant tumors in western countries.The incidence of PCa in Asian countries is increasing in recent years Androgen deprivation therapy(ADT)is an effective treatment for newly diagnosed PCa.Unfortunately,ADT failure can lead to castration-resistant prostate cancer(CRPC),which shows more aggressive and poor prognosis.Therefore,CRPC is one of the hot issues in the current research,but its pathogenesis is not clear.Thus,a better understanding of the molecular events in androgen-independent(AI)PCa cells may aid in the development of novel targets to improve CRPC patients' outcomes.There are AR dependent mechanism and AR independent mechanism for CRPC development.The roles of miRNA,mismatch repair protein defects,autophagy and prostate stem cells in CRPC have also been reportedMiRNA is a small molecule non-coding conservative single-stranded RNA.MiRNA can target the target gene by complete or incomplete complementary binding with mRNA 3'UTR,which cause the degradation of mRNA or inhibit its normal translation.MiRNA plays an important role in the process of cell metabolism,proliferation,differentiation,cell death and cell survival.Because of these important biological functions,miRNA is considered to play an important role in carcinogenesis.The expression of miRNA in PCa is becoming more and more important because of its application in diagnosis,stage,prognosis prediction and treatment.MiR-30a is a member of the miR-30 family,most of which are low-expression in malignant tumors and play the role of tumor suppressor genes.MiR-30a has been reported to be down-regulated in lung cancer,breast cancer,renal cell carcinoma,hepatocellular carcinoma,colorectal cancer,prostate cancer,gastric cancer and other malignant tumors.MiR-30a plays an important role in cancer by regulating target genes,which inhibit proliferation,invasion and migration,and induce apoptosis.In addition,miR-30a increases the sensitivity of gastric cancer cells to cisplatin by inhibiting epithelial to mesenchymal transformation of(EMT).Therefore,miR-30a may become a potential target for tumor therapy.MiR-30a has been reported to be related to the proliferation,invasion,migration,EMT and radiosensitivity of PCa cells.However,the role and mechanism of miR-30a in CRPC is not clear till now.In this study,bioinformatics was used to analyze the expression and significance of miR-30a in localized PCa and CRPC,and the role and mechanism of miR-30a in AI proliferation of PCa cells was exploredPart ? The expression and clinical significance of miR-30a in PCa PCa,as a malignant tumor with high incidence in men,often transforms into CRPC after ADT failure.The pathogenesis of CRPC is not clear.There are AR dependent mechanism and AR independent mechanism in the development of CRPC.The role of miRNAs in localized PCa and CRPC has also been studied.In this study,bioinfonnatics methods were used to analyze the expression of miRNA in multiple public databases of PCa.1.The expression of 6 miRNAs are downregulation in CRPC compared with benign prostate tissue and localized PCa:Meta-analysis of 10 GEO data sets showed that there was 101 differential expression miRNAs between benign prostate tissue and localized prostate cancer tissue,of which 80 miRNAs were down-regulated and 21 miRNAs were up-regulated.After cluster analysis of these 101 miRNAs expression in TCGA database,it was found that this group of miRNAs expression profiles can accurately distinguish benign prostate tissue from localized PCa tissue After converging 80 down-regulation miRNAs and the differential miRNAs between localized PCa and CRPC in the two datasets(GSE80400 and Wang et al,32 fold change?2),we found six candidate miRNAs:miR-30a,miR-99a,miR-100,miR-205,miR-27b and miR-143.The expression of 6 miRNAs are downregulation in CRPC compared with benign prostate tissue and localized PCa.2.The down-regulation expression of miR-30a in prostate cancer indicates a poor prognosis:TCGA data analysis showed the down-regulation of miR-30a was most significantly associated with poor biochemical recurrence(BCR)free survival among 6 miRNAs.The down-regulation of miR-30a is associated with BCR,high T stage,high Gleason grade group of PCa.Gene Set Enrichment Analysis(GSEA)analysis showed that miR-30a-downregulated gene signatures were significantly enriched in CRPC phenotypc.Part ? MiR-30a inhibits androgen-independent proliferation of PCaIn this part,we studied the role and possible signal pathways of miR-30a on PCa AI growth by bioinformatics analysis and experiments in vivo and in vitro1.The expression of miR-30a in PCa cell line:RT-qPCR analysis showed that the basic expression level of miR-30a in VCap,LNCaP,PC3,22RV1 and DU145 decreased gradually.We chose androgen-dependent cell line LNCaP and AI cell line 22RV1 to investigate the role of miR-30a.2.MiR-30a could inhibit androgen independent proliferation of PCa cells in vitro:After androgen deprivation,MTS,EdU and colony formation assay showed that over-expression of miR-30a could inhibit the growth and colony formation of 22RV1 cells,while inhibition of miR-30a expression could improve the growth and colony formation of LNCaP cells.Exogenous DHT stimulation reduced the inhibitory effect of miR-30a on the growth of PCa.3.MiR-30a could inhibit the tumorigenesis of nude mice:22RV1 cells were lentivirally transduced to stably express miR-30a or negative control and were subcutaneously implanted into the axilla of nude mice that have undergone bilateral orchiectomy.The 22RV1 cells with miR-30a overexpression displayed a delay in palpable tumor onset and a significant decrease in tumor growth and weight when compared with the controls(P<0.05).4.MiR-30a could affect the expression of cell cycle related genes:GO and KEGG analysis of the differentially expressed genes between miR-30a high expression group and low expression group in TCGA showed that they were closely related to cell cycle.Flow cytometry analysis showed that overexpression of miR-30a in 22RV1 cells inhibited G1/S phase transition,while inhibition of miR-30a expression in LNCaP cells promoted G1/S phase transition.There was a significant difference in the expression of cell cycle control genes between the low-and high-expression samples of miR-30a in TCGA.Further RT-qPCR and immunohistochemical analysis confirmed that miR-30a could affect the expression of cell cycle related genes and proteins.5.MiR-30a could affect the expression of AR-mediated transcription genes:AR-mediated transcription genes also showed differential expression between the low-and high-expression tumors of miR-30a in TCGA.RT-qPCR analysis showed that miR-30a inhibitor could reduce the stimulating effect of exogenous DHT on the expression of PSA and TMPRSS2 in LNCaP cells and increase the expression level of CDC20,while miR-30a mimics could enhance the stimulating effect of exogenous DHT on the expression of PSA and TMPRSS2 in 22RV1 cells and inhibit the expression level of CDC20.These results suggest that miR-30a could alter AR-mediated transcription genes.6.The correlation between miR-30a and AR mediated transcription genes or cell cycle related genes:We further observed whether there was a linear correlation between miR-30a and AR mediated transcription genes or cell cycle related genes in tissue samples of TCGA.The correlation analysis showed that miR-30a was positively correlated with AR mediated transcription genes KLK2,KLK3,KLK4 and TMPRSS2(P<0.01),and negatively correlated with cell cycle related genes MDM2,GSK3B,MKI67,CDC20 and CCNB1(P<0.01).Part ? Expression of miR-30a target genes SOX4,MYBL2 and FOXD1 in prostate cancer and their effects on proliferationIn this part,MYBL2 and FOXD1,which play important roles in cell proliferation,were screened and identified as miR-30a target genes by bioinformatics and molecular biology experiments.Combined with SOX4 we have previously found,we explored the expression and clinical significance of these three miR-30a target genes in prostate cancer,and their role in PCa AI growth.1.MiR-30a directly targets MYBL2 and FOXD1:Bioinformatics analysis tools predict that MYBL2 and FOXD1 may be target genes of miR-30a.The results of Western blot showed that when miR-30a mimic was transfected into LNCaP and 22RV1 cells,the protein expression of MYBL2 and FOXD1 was significantly decreased compared with the control group,while the expression of MYBL2 and FOXD1 was significantly increased when miR-30a inhibitor was transfected into LNCaP and 22RV1 cells.Dual-luciferase assay showed that miR-30a mimics could inhibit the 3'untranslated region activity of MYBL2 and FOXD1.2.The correlation analysis between miR-30a and SOX4,MYBL2 and FOXD1 in TCGA:The correlation analysis between miR-30a and SOX4,MYBL2 and FOXD1 in TCGA showed that there was a negative correlation between miR-30a and the three target genes.We further analyzed the expression correlation between miR-30a signature genes and SOX4,MYBL2 and FOXD1 signature genes.There was also a negative correlation between them.3.The expression and significance of SOX4,MYBL2 and FOXD1 in PCa:The expression of SOX4,MYBL2 and FOXD1 showed a pairwise positive correlation in GSE35988(r=0,40-0.59).The expression of SOX4,MYBL2 and FOXD1 increased gradually in benign prostate tissue,localized PCa and CRPC.Principal component analysis showed that the signature gene expression profiles of SOX4,MYBL2 and FOXD1 could effectively distinguish localized prostate cancer from CRPC.Over-expression of SOX4,MYBL2 and FOXD1 suggested poor BCR-free survival in TCGA,respectively.4.SOX4,MYBL2 and FOXD1 could promote PCa AI growth:MTS experiments showed that siRNAs of SOX4,MYBL2 and FOXD1 could inhibit the proliferation activity of 22RV1 cells in androgen deprivation environment.In addition,miR-30a inhibitor could increase the proliferation of LNCaP cells,but the siRNAs of SOX4,MYBL2 and FOXD1 could partially reverse the above effects.5.Effects of SOX4,MYBL2 and FOXD1 on cell cycle related genes and AR-mediated transcription genes:We detected the expression of 8 cell cycle related genes by RT-PCR.The results showed that after transfection of SOX4,MYBL2 and FOXD1 siRNA,the mRNA expression of CDC20,CCNB1,CDK4,E2F1,GSK3B,MKI67 and MDM2 decreased,while the mRNA expression of FZR1 increased.SiRNAs of SOX4,MYBL2 and FOXD1 slightly increased the expression of PSA and TMPRSS2 genes and decreased the expression level of CDC20 in 22RV1,while when further DHT stimulation,the expression levels of PSA and TMPRSS2 were significantly increased and the expression levels of CDC20 decreased more significantly.MiR-30a inhibitor decreased the expression of PSA and TMPRSS2 and increased the expression of CDC20 in LNCaP,but the above effects were weakened after co-transfection of miR-30a inhibitor and siRNA of SOX4,MYBL2 or FOXD1.In summary,our study found that the expression of miR-30a are downregulation in CRPC compared with benign prostate tissue and localized PCa,and was related to clinical stage,Gleason grade and BCR.Low-expression of miR-30a and over-expression of target genes were poor prognostic factors.MiR-30a could inhibit PCa AI growth by directly targeting SOX4,MYBL2 and FOXD1,which may be related to regulating the expression of cell cycle-related genes and AR-related pathway genes in PCa cells.These results will provide further theoretical basis for the choice of therapeutic strategies and the design of new therapeutic targets for PCa including CPRC.