Androgen Receptor (AR) Promotescell Proliferation And Metastasis By Suppressing MiR-145 In Renal Cell Carcinoma (RCC)

Part ? AR promotes invasion and proliferation of RCC cellObjectiveTo study the function of AR in various RCC cell lines.MethodsWestern blot analysis for the expression of AR in OSRC-2, HK2, ACHN and SW-839 cells. To confirme whether ARpromotes invasion and proliferation of RCC cell, we modulated the levels of AR in RCC cells with either a shRNA or a vector encoding AR and established stable RCC cell lines by lentiviral transduction. To detect the effect of transduction, we performed western blot analysis. To evaluate whether AR promotes the invasion of RCC cells, we performed matrigel-coated transwell invasion assay andanother 3D culture invasion assay. Next,we also examined the effects of differential AR expression on RCC cell proliferation using MTT assays.ResultsWestern blot analysis showed thatAR is highly expressed in SW-839 cells while lowly expressed in ACHN and OSRC-2 cells. Western blot analysis revealed that the regulation of AR expression was successful in different RCC cell lines.Using matrigel-coated transwell invasion assay, we found knocking-down AR in SW-839 cells suppressed cell invasion. In contrast, exogenous expression(OE) of AR in OSRC-2 cells ACHN cells resulted in increased cell invasion compared with the control cells.Similar results were revealed in 3D culture invasion assay.MTT assay revealed that knocking-down AR in SW-839 cells led to slower cell proliferation while addition of AR in OSRC-2 cells and ACHN cells resulted in increased cell proliferation.ConclusionsAR promote RCC cell invasion and proliferation.Part ?AR promotes RCC cell invasion and proliferation via regulation of miR-145 expressionObjectiveTo explore the mechanism by which AR promotes RCC cell invasion and proliferation and explore whether miR-145 is involved in AR-enhanced RCCprogression.MethodsAn array of miRNAs which are related to RCC invasion and proliferation was applied to found which miRN A was the most significantly suppressed by AR.Next, to evaluate the effect of AR on miR-145 expression, we performed Real-time PCR after modulated the levels of AR in RCC cells. In order to further confirm the conclusion, we performed Real-time PCR to examined the AR/miR-145 expression in human RCC samples.Transwell assay and MTT assays were performed after regulating AR/miR-145 expression to explore whether miR-145 is involved in AR-enhanced RCC progression.ResultsMiRNA-145 was the most significantly suppressed by AR in the array of miRNAs. Real-time PCR analysis revealed that shRNA-mediated knowdown of AR in SW-839 cells induced miR-145 levels, whereas overexpression of AR in OSRC-2 and ACHN cells significantly reduced miR-145 levels.To furtherconfirm this finding, we then examined the AR/miR-145 levels in human RCC samples. Takentogether, results from above suggested that AR could reduce miR-145 expression in human RCC.Transwell assay and MTT assays showed that AR can function through suppression of the miR-145 expression to promote RCC progression.ConclusionsAR suppresses miRNA-145 expression in human RCC.AR enhances RCC cell invasion and proliferation via suppression of miR-145 expression.Part?AR-suppressed miR-145 enhances the HIF2a/VEGF/MMP9/CCND1 signals promote RCC progressionObjectiveTo explore the mechanism how AR suppress miR-145expression at the molecular level and explore how AR-suppressed miR-145 signals enhance the RCC progression.MethodsTo predict the putative AR response elements on the miR-145 promoter region, we applied PROMO virtual lab. In order to further determine whether the AR might bind to the ARE located at miR-145 promoter region, chromatin immunoprecipitation (ChIP) assays was performed in RCC cells using PCR primers covering the sequence of interest.To explore whether AR might suppress miR-145 expression function through p53, We therefore performed Real-time PCR analysis to detect the expression ofsh-AR-induced miR-145 when we knocked-down p53 by p53-shRNA. We performed Western blot analysis to detect the expressionof HIF2a/VEGF/ MMP9/CCND1 compared to their controls after we knocked-down AR in SW-839 cellsandoverexpressed AR in OSRC-2 cells and ACHN cells.To evaluate the effect of miR-145 on HIF2a/VEGF/MMP9/CCND1, we performed Western blot analysis. We performed Western blot analysis to detect the expression of HIF2?/VEGF/MMP9/CCND1 bytransfecting miR-145 inhibitor (inn) or miR-145 mimic into sh-AR SW-839 cells or OE-AR OSRC-2 cells. We then examin the impact of miR-145 mimic on VHL in ACHN cells with wild-type VHL and in OE-AR ACHN cells with wild-type VHL.ResultsARE locate at 575 bp upstream to the transcriptional start site of the human pre-miR-145 and ChIP assays revealed that AR could bind to the AREsuggesting AR might suppress miR-145 expression at the transcriptional level. Real-time PCR analysis showed thatmiR-145 expression induced by sh-AR was significantly suppressed when p53 was knocked-down by p53-shRNA.Knocking-down AR in SW-839 cells suppressed the expression of HIF2a/VEGF/MMP9/CCND1.In contrast, addition of AR in OSRC-2 cells and ACHN cells resulted in enhanced expression of HIF2a/VEGF/MMP9/CCND1.Transfecting miRNA-145 mimic also led to suppress the expression of HIF2o/VEGF/MMP9/CCND1 in SW-839 and OSRC-2 cells and the interruption approach transfecting miR-145 inhibitor or miR-145 mimic could partially reverse the AR effect on the expression of HIF2a/VEGF/MMP9/CCND1 signals.MiR-145 alone could suppress VHL expression in ACHN cells with wild-type VHL. Importantly, adding miR-145 could reverse the AR-enhanced VHL expression.AR-suppressed miR-145 signals can override the opposing AR-enhanced VHL signal to result in the increase of the HIF2a/VEGF/MMP9/CCND1 expression in VHL wild-type RCC ACHN cells.ConclusionsAR suppresses miR-145 promoter activityvia regulation of p53.AR-suppressed miR-145 regulates the HIF2a/VEGF/MMP9/CCND1 signals play key roles to promote RCC progression.Part?AR-suppressed miR-145 enhances the HIF2a/VEGF/MMP9/CCND1 signals promote RCC progression in vivoObjectiveTo prove the above data in vivo.MethodsWe modulated the levels of AR/miR-145 in RCC cells with a vector encoding AR/miR-145 and established stable RCC cell lines by lentiviral transduction. To detect the effect of transduction, we performed QPCR.Orthotopically inplanted these RCC cells under the kidney capsules of nude mice. The RCC growth and metastases were monitored by IVIS imaging system 5 weeks after implantation.Then the nude mice were sacrificed and the tumor sizes in each mouse weremeasured and weighed.The metastatic tumors in the hepatic hilar region and diaphragm were observed and confirmed by H&E staining.IHC staining confirmed the expression of AR/HIF2a/VEGF/MMP9/CCND1 in different mice.ResultsThe results revealed that adding AR in OSRC-2 cells increased tumor growth and weight in nude mice compared to Vec group, while adding miR-145 in the OE-AR OSRC-2 cells could partially reverse the impact of AR and decreased the RCC tumor growth and weight.We also found increased metastatic foci in mice with OE AR RCC cells and no metastatic foci in mice with OE miR-145 RCC cells. Metastatic tumor cells in the hepatic hilar region and diaphragm were confirmed to be RCC cells by H&E staining.IHC staining confirmed the expression of AR/HIF2a/VEGF/MMP9/ CCND1 in vivo is consistent with in vitro results.ConclusionsAR-suppressed miR-145 enhances the HIF2?/VEGF/MMP9/CCND1 signals promote RCC progression in vivo.