Mechanism Of MiR-543 Regulating Proliferation And Invasion Of Renal Clear Cell Carcinoma By Targeting KLF6

Objective:Clear cell renal cell carcinoma(ccRCC)is a urinary disease with high incidence and the incidence is only lower than bladder cancer in urological cancers.The incidence of ccRCC is increasing every year in China.To date,the molecular pathogenesis of ccRCC remains poorly understood.Therefore,it's important to explore the mechanism underlying the development and progression of ccRCC,and provide novel targets for the development of therapeutic strategy of ccRCC.Increasing studies have shown that microRNA-543(miR-543)is dysregulated in various cancers,including colorectal cancer,breast cancer and prostate cancer.miR-543 is reported to participate in the development and progression of cancers through regulating the expression of target genes.The Krupple-like factor 6(KLF6)gene is closely related to tumorigenesis and progression.Studies have shown that KLF6 is dysregulated in ccRCC,and is involved in the development and progression of ccRCC.Bioformatic analysis predicts that KLF63'-untranslated region(3'-UTR)contains potential binding sites for miR-543.However,whether miR-543 regulates the development and progression of ccRCC by targeting the3'-UTR of KLF6 mRNA remains unclear.This study aimed to investigate the expression pattern of miR-543 in ccRCC tissues and cells and explore the role of miR-543 in regulating the proliferation and invasion of ccRCC cells.Addtionally,this study aimed to elucidate the regulatory effect of miR-543 on KLF6 and the underlying molecular mechanism on regulating ccRCC cell proliferation and invasion.This study will provide novel insights into understanding the molecular pathogenesis of ccRCC and suggest potential targets for the drug development and clinical treatment of ccRCC.Methods:1.The expression of miR-543 in ccRCC tissues and cell lines was detected by RT-qPCR;Overexpression of miR-543 was achieved by transfecting miR-543 mimics into ccRCC cells;inhibition of miR-543 was achieved by transfecting miR-543 inhibitor into ccRCC cells;The effect of miR-543 on regulating ccRCC cell proliferation,cell cycle and apoptosis was detected by CCK-8 or flow cytometry assays;the effect of miR-543 on regulating ccRCC cell invasion was detected by Transwell invasion assay;2.Bioinformatic analysis and dual-luciferase reporter assay were performed to investigate the targeting relationship between miR-543 and 3'-UTR of KLF6 mRNA;The effect of miR-543 on the expression of KLF6 and p21 was detected by RT-qPCR and Western blot;The correlation of miR-543 and KLF6 expression in ccRCC tissues was analyzed by Spearman's rank correlation analysis;3.The functional regulation between miR-543 and KLF6 was validated by cotransfection of miR-543 mimics and KLF6 expression vector or miR-543 inhibitor and KLF6 siRNA into ccRCC cells.Results:1.The expression of miR-543 was significantly increased in ccRCC tissues and cell lines;Overexpression of miR-543 could promote the proliferation and invasion of ccRCC cells,and reduce the proportion of cells in G0/G1 phase and the rate of apoptosis;inhibiton of miR-543 showed opposite effect;2.Bioinformatics analysis predicted that the KLF6 3'-UTR contained potential targeting sites for miR-543.Dual-luciferase reporter assay confirmed that miR-543 directly targeted KLF6 3'-UTR;Overexpression of miR-543 inhibited KLF6 expression while inhibition of miR-543 promoted KLF6 expression.Addtionally,an inverse correlation of miR-543 and KLF6 expression was evidenced in clinical ccRCC specimens;miR-543 negatively regulated p21 expression through targeting KLF6;3.Overexpression of KLF6 could reverse the regulatory effect of miR-543 overexpression on the biological function of ccRCC cells;whereas silencing of KLF6 could reverse the regulatory effect of miR-543 inhibition on the biological function of ccRCC cells.Conclusions:High expression of miR-543 in ccRCC tissues and cells indicates a oncogenic role of miR-543 in ccRCC;miR-543 promoted the proliferation and invasion of ccRCC cells;miR-543 inhibited the expression of KLF6 through targeting the 3'-UTR of KLF6;miR-543 regulated the expression of p21 by targeting KLF6;miR-543 regulated the proliferation and invasion of ccRCC cells through targeting KLF6,and thus participated in the development and progression of ccRCC.This study provides a new theoretical basis for elucidating the molecular mechanism underlying the development and progression of ccRCC.Moreover,this study provides novel and promising targets for the development of therapeutic drugs and clinical treatment for ccRCC.