The Study Of Deregulated MicroRNA-503 Mediated ZNF217 Expression In Prostate Cancer

Objective:Prostate cancer is one of the most common cancers among men with high incidence and mortality worldwide.Although increasing evidence demonstrated that deregulation of mircoRNA-503(miRNA-503)contributes to tumorigenesis,little is known about the biological role and intrinsic regulatory mechanisms of miR-503 in prostate cancer.To detect the expression of miR-503 in prostate cancer tissues and different cell lines,investigate the effect of miR-503 on the biological behavior of prostate cancer in vitro and in vivo,and explore the biological mechanism of miR-503 mediated prostate cancer progression.Methods:The expression of mi R-503 in different cell lines of prostate cancer and prostate cancer was detected by quantitative real-time PCR(qRT-PCR).To further explore its biological role in prostate cancer,we thereafter stably overexpression of miR-503 in PC3 cells with specific lentiviral vector.Cell growth,tumor formation,cell invasion and migration were detected by CCK-8 assay,colony formation assay and transwell assay,respectively.To elucidate the underlying mechanism by which miR-503 exerts its function in prostate cancer cells,we searched miR-503 target genes using an online software.ZNF217 was identified as a potential target of miR-503 based on putative target sequences.The luciferase assay,qRT-PCR and Western blot were performed to examine the post-regulation of ZNF217 by miR-503.To further clarify the contribution of ZNF217 to prostate cancer progression,we rigorously examined the functional involvement of ZNF217 in rescue experiments.Cell proliferation,colony formation,tumor invasion and migration were also detected upon ZNF217 knockdown or overexpression.In addition,mechanistic studies revealed abnormal expression of ZNF217 on epithelial-mesenchymal transition and iron metabolism-related genes(such as E-cadherin and Ferroportin)by RT-PCR,Western blot,luciferase assay and chromatin imunoprecipitation(ChIP)assay.Besides,to explore how miR-503 expression was downregulated in prostate cancer,wehypothesized that unknown transcriptional factors might regulate miR-503 expression.Based on the bioinformatics analysis,we found that the promoter of mature miR-503 contains a putative GATA3 binding site.The luciferase assay and ChIP assay were performed to determine the transcriptional regulation of mature miR-503 by GATA3.Cell proliferation,colony formation,tumor invasion and migration were also detected upon GATA3 knockdown or overexpression.In addition,RNA immunoprecipitation assay and luciferase assay were performed to detect the competitively binding of miR-503 by long chain non-coding RNA(lncRNA)NEAT1.Finally,we also detected the mRNA levels of GATA3,lncRNA-NEAT1,ZNF217,Ferroportin in prostate cancer tissues by RT-PCR.Spearman correlation analysis was used to calculate the correlation between miR-503 and other clinical factors.The Kaplan-Meier method tests were utilized for survival analysis.Results:In present study,we found that miR-503 was significantly downregulated in advanced prostate cancer tissues and cell lines.Downregulation of mi R-503 was strongly associated with aggressive clinical-pathological features and poor prognosis in PCa patients.Ectopic expression of mi R-503 significantly inhibited tumor cells growth,cell migration and invasion in vitro and in vivo.Mechanistic studies revealed that ZNF217 was a direct target downstream target of mi R-503.Knockdown of ZNF217 mimicked the tumor-suppressive effects of miR-503 overexpression on prostate cancer invasion,whereas ZNF217 overexpression attenuated the tumor-suppressive function of miR-503.Subsequently,miR-503 further modulated the activation of ZNF217-downstream E-cadherin and ferroportin expression,thus led to epithelial-mesenchymal transition processes and iron egress,respectively.Importantly,we also found that GATA3 transcriptionally modulated miR-503 expression,and lncRNA-NEAT1 was also involved in regulating ZNF217 expression through competitively ‘sponging' miRNA-503.These results correlate with reduced GATA3 and elevated lncRNA-NEAT1 expression found in advanced prostate cancer tissues that result in diminished miRNA-503 levels.Conclusion:Genetic and epigenetic silencing of miR-503 enhances ZNF217 oncogene expression to foster prostate cancer progression.Targeting molecules within the miR-503-ZNF217 signaling thus appears to be a promising approach to restrain prostate cancer progression.