下调miR-3613-5p通过靶向FRMD6调控肾癌细胞增殖和细胞周期

Down-regulation of miR-3613-5p regulates renal cancer cell proliferation and cell cycle by targeting FRMD6

目的探讨下调miR-3613-5p通过靶向含FERM结构域蛋白6(FRMD6)调控肾癌细胞增殖和细胞周期的分子机制。方法以OncoLnc预后分析数据库分析miR-3613-5p表达水平与肾癌患者预后的关系。用qPCR检测肾癌细胞株(A498、Caki-1、OS-RC-2、ACHN)和正常肾小管上皮细胞HK-2中miR-3613-5p的表达水平。分别将miR-3613-5p抑制物和阴性对照质粒(si-miRCon)转染至Caki-1细胞,分为si-miR-3613-5p组和si-miR-Con组。用CCK-8法和PI染色流式细胞术分别检测miR-3613-5p抑制物对Caki-1细胞增殖和细胞周期的影响。qPCR和Western blotting检测Caki-1细胞中FRMD6 mRNA和蛋白的表达。Western blotting检测AKT/MAPK信号通路蛋白的表达。双荧光素酶报告基因实验验证miR-3613-5p和FRMD6的靶向关系。结果与miR-3613-5p表达较高的肾癌患者相比,miR-3613-5p低表达肾癌患者的生存率较高(P<0.01)。与HK-2细胞相比,miR-3613-5p在肾癌细胞株中高表达(均P<0.05),Caki-1细胞中miR-3613-5p表达最高(P<0.01)。与si-miR-Con组相比,si-miR-3613-5p组Caki-1细胞增殖能力显著被抑制(P<0.05),细胞周期进展被明显抑制(P<0.01),FRMD6基因表达明显增加(P<0.01),AKT/MAPK信号通路蛋白p-AKT1、p-MAPK3、CCND1表达明显降低。双荧光素酶报告基因实验证实FRMD6是miR-3613-5p的靶基因。结论下调miR-3613-5p表达能够明显促进FRMD6基因表达,进而抑制肾癌细胞增殖和细胞周期进展。

Objective To explore the effects of down-regulation of miR-3613-5p on the proliferation and cell cycle of renal cancer cells by targeting FERM domain-containing protein 6(FRMD6)and its molecular mechanism.Methods The OncoLnc prognostic analysis database was used to analyze the relationship between the expression level of miR-3613-5p and the prognosis of renal cancer patients.Fluorescence quantitative polymerase chain reaction(qPCR)was used to detect miR-3613-5p expression level in renal cancer cell lines(A498,Caki-1,OS-RC-2,ACHN)and normal renal tubular epithelial cells HK-2.The miR-3613-5p inhibitor and the negative control plasmid(simiR-Con)were respectively transfected into Caki-1 cells,namely the si-miR-3613-5p group and the si-miR-Con group.CCK-8 method and PI staining flow cytometry were used to detect the effects of miR-3613-5p inhibitor on the proliferation and cell cycle of Caki-1 cells.Western blotting and qPCR were used to detect FRMD6 mRNA and protein expression in Caki-1 cells.Western blotting was used to detect the expression of AKT/MAPK signaling pathway proteins.The dual luciferase reporter gene experiment verified the targeting relationship between miR-3613-5p and FRMD6.Results Compared with renal cancer patients with high miR-3613-5p expression,renal cancer patients with low miR-3613-5p expression had a higher survival rate(P<0.01).Compared with HK-2 cells,the expression of miR-3613-5p was high in renal cancer cell lines(all P<0.05),and the expression of miR-3613-5p was the high-est in Caki-1 cells(P<0.01).Compared with the si-miR-Con group,the proliferation ability of Caki-1 cells in the si-miR-3613-5p group was significantly inhibited(P<0.05),and the cell cycle progression was significantly inhibited(P<0.01).The FRMD6 gene expression in Caki-1 cells in the si-miR-3613-5p group was significantly increased(P<0.01),and the ex-pression of AKT/MAPK signaling pathway proteins p-AKT1,p-MAPK3,CCND1 was significantly reduced.The dual lucifer-ase reporter gene experiment confirmed that miR-3613-5p targets FRMD6.Conclusions Down-regulation of miR-3613-5p expression can significantly promote FRMD6 gene expression,thereby inhibiting renal cancer cell proliferation and cell cycle progression.