| Prostate cancer(PCa)is the most common malignancy among men in the western countries.In recent years,with the economy development,the incidence and mortality rates of PCa have also been steadily elevated in China.PCa has become one of the major diseases that threaten the health of Chinese men.Non-coding RNAs including microRNAs and lncRNAs have been widely studied in the area of PCa progression and metastasis.It has recently become apparent that there is interesting cross-regulation between miRNAs and lncRNAs during malignant process of tumors.However,the cross-regulation between miRNAs and lncRNAs has not been systematically investigated in the progression of prostate cancer.Accumulating evidence indicates that HOTAIR is highly expressed in a variety of cancer types and serves as an oncogene in the malignant process of various cancers including prostate cancer.Recent study indicated that HOTAIR could directly bind to the AR protein to prevent its ubiquitination and degradation,thereby promoting PCa cell growth and invasion.Another important mechanism is that HOTAIR could recruit polycomb repressor complex 2(PRC2)which methylates lysine27 of histone H3(H3K27)to promote transcriptional silencing of many tumor suppressive genes.Nevertheless,the role of cross-regulation between HOTAIR and miRNA in PCa progression has not been reported.In our previous microarray analysis,we have detected a cluster of miRNAs which were downregulated in CRPC compared with that in ADPC(androgen dependent prostate cancer).Furthermore,we reanalyzed the GEO dataset(GSE26996)to identify EZH2 negatively-regulated miRNAs in DU145.In addition,to reveal the potential miRNAs that can regulate HOTAIR in prostate cancer,we searched miRcode algorithm,DIANA Tools and starBase v2.0.A disciplinary overlap was showed by intersecting these three groups of miRNAs.16 miRNAs were shared.By aligning these miRNAs to MSKCC prostate cancer dataset(GSE21032),microRNA-193a-3p(miR-193a)was found to be one of the most significantly downregulated miRNAs in metastatic PCa tissues compared with primary cancer.However,the exact biological function of miR-193 a in tumorigenesis of PCa remains largely unknown.In the present study,we retrieved and reanalyzed the original miRNAs expression and clinical data from the Memorial Sloan Kettering Cancer Center(MSKCC)and The Cancer Genome Atlas(TCGA)databases to investigate clinical relevance of miR-193 a with the pathological traits of patients.Prostate cancer cell functional experiment was further performed to verify the effect of EZH2/miR-193 a biological axis on prostate cancer cells.Moreover,we explored that EZH2 coupled with HOTAIR to silence miR-193 a through introducing trimethylation of H3K27 at miR-193 a promoter region by using molecular biology experiments.Finally,we proved that HOTAIR was a direct target of miR-193 a in PCa by using bioinformatics and molecular biology techniques.This study aims to investigate the role of HOTAIR/EZH2/miR-193 a feedback loop in the progression of PCa which could provide solid theoretical basis for target therapy and early detection of PCa.In the first part,MSKCC data indicated that the expression of miR-193 a in metastatic cancer was significantly decreased compared to primary cancer.We also found that miR-193 a expression was significantly downregulated in higher-T stage tumors via reanalysis of TCGA dataset.We evaluated the miR-193 a expression in our clinical specimens via ISH(in situ hybridization)and found that miR-193 a was aberrantly downregulated in high-Gleason score tumors(Gleason score: 8–10).CCK-8 and colony formation assays indicated that miR-193 a significantly alleviated the promoting effect of EZH2 on cell growth.Conversely,anti-miR-193a(miR-193 a inhibitor)could also partially abrogate the inhibition effect of proliferation caused by knock-down of EZH2 in PCa cells.GSEA showed that a negatively enriched expression of gene sets was involved in mitotic spindle assembly and G2/M cell-cycle checkpoint in miR-193a-overexpressing cells.EZH2 depletion increased the apoptosis rate in both PC3 and DU145 cells which could be partially rescued by supplementing the cells with anti-miR-193 a.On the other hand,upregulation of miR-193 a in EZH2-overexpressing cells could significantly induce apoptosis in PCa cells.These results were both confirmed by flow cytometry and TUNEL assay.Transwell assay indicated that overexpression of miR-193 a in PCa cell lines strongly suppressed cell migration and invasion abilities.GSEA showed that a negatively enriched expression of genes sets was involved in hallmarks of TGF-? signaling,TNF-? via NF-kB signaling and KRAS prostate up signaling in miR-193a-overexpressing PCa cells.Overexpression of miR-193 a via using lentivirus obviously suppressed tumor growth as manifested by reduced tumor size and tumor weight.Immunohistochemical staining(IHC)illustrated that reduced Ki67-positive cells,CD31-positive cells and CD34-positive cells were found in miR-193a-overexpressed xenograft tumor cells.These results indicated that miR-193 a serves as a tumor suppressor via inhibiting PCa cells proliferation,migration,invasion and promoting cell apoptosis in PCa,which could partially abrogate the oncogenic function of EZH2.Though the expression profile and biological function of miR-193 a have been illustrated,the molecular mechanisms for regulation of miR-193 a expression in prostate cancer are not clear.In second part,in order to prove that EZH2 had a direct role in repressing miR-193 a in PC3 and DU145,we depleted EZH2 by stably infection of lentiviral particles of LV-shEZH2 or upregulated EZH2 with plasmid pcDNA3.1-EZH2 in PCa cells,and qRT-PCR was used to measure the level of miR-193 a.To prove that HOTAIR was important in EZH2 mediated silencing of miR-193 a,we inhibited the HOTAIR expression by siRNAs in EZH2-overexpressing PCa cells and then measured miR-193 a expression level.Results indicated that there were upregulation of miR-193 a expression in PC3 and DU145 cells upon inhibition of EZH2 or HOTAIR.And ectopic expression of EZH2 could significantly reduce the level of miR-193 a expression.Western blot assay showed that the enrichment of H3K27me3 significantly decreased when EZH2 was knocked down in LV-shEZH2 infected PCa cells and vice versa.WB also indicated that both expression of EZH2 and H3K27me3 largely declined in the HOTAIR knocking down PCa cells.We cloned 2.0 kb fragments upstream of TSS(transcription starting site)of human pri-miR-193 a stem-loop into pGL3-basic-vector to generate luciferase construct pGL3-193a-promoter reporter.Luciferase assay and ChIP assay clearly suggested that EZH2 coupled with HOTAIR to induce H3K27 trimethylation at miR-193 a promoter,which reduced miR-193 a expression in PC3 and DU145 cells.In third part,qRT-PCR was used to measure the HOTAIR expression in PCa cells either via transfecting with miR-193 a mimics or infecting with LV-miR-193 a and in xenograft tumor tissues which were generated from PC3 cells stably overexpressing miR-193 a.We observed ectopic miR-193 a expression significantly reduced HOTAIR level both in PCa cells and PCa tumor tissues.The luciferase reporter assay demonstrated that miR-193 a significantly reduced luciferase activity(wild type).Mutation of miR-193 a binding site in HOTAIR abrogated the inhibitory effects.Spearman correlation analysis showed significant inverse correlation between expression of HOTAIR and miR-193 a as well as between that of EZH2 and miR-193a(P < 0.001)in 31 PCa specimens.Thus,miR-193 a directly targets HOTAIR and negatively modulates HOTAIR expression in prostate cancer.As miR-193 a was epigenetic silenced by EZH2 and HOTAIR,in return the suppression effect of HOTAIR by miR-193 a was largely abrogated.Thus,the tumor suppressor miR-193 a was continuously inhibited and oncogene HOTAIR remained highly expressed in prostate cancer,which promoting initial prostate cancer developed into highly aggressive cancer type. |
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