circPVT1靶向调控miR-195-5p/SOX9轴影响结肠癌细胞增殖、凋亡及上皮间质转化

circPVT1 targets miR-195-5p/SOX9 axis to affect proliferation,apoptosis and epithelial-mesenchymal transformation of colon cancer cells

目的:探究环状RNA浆细胞瘤变体易位1(circPVT1)靶向调控微小RNA-195-5p(miR-195-5p)/性别决定区Y框蛋白9(SOX9)轴对结肠癌细胞增殖、凋亡及上皮间质转化(EMT)的影响。方法:体外培养人正常结肠上皮细胞NCM-460、人结肠癌细胞系(SW480、HCT116、HCT29、LOVO、SW620)至对数期,将SW480细胞分为对照组(正常培养SW480细胞不进行转染)、空载质粒组(转染空载质粒)、circPVT1沉默组[转染circPVT1小干扰RNA(siRNA)质粒]、miR-195-5p过表达组[转染miR-195-5p模拟物(mimics)质粒]、SOX9沉默组(转染SOX9 siRNA质粒)、circPVT1沉默+miR-195-5p低表达组(转染circPVT1 siRNA和miR-195-5p siRNA质粒)。各组转染相应质粒后培养48 h。荧光定量PCR法检测NCM-460细胞、人结肠癌细胞系(SW480、HCT116、HCT29、LOVO、SW620)和各组SW480细胞circPVT1、miR-195-5p、SOX9 mRNA的表达;噻唑蓝和流式细胞仪分别检测各组SW480细胞增殖和凋亡;蛋白印迹法检测各组SW480细胞SOX9、凋亡和EMT相关蛋白表达;双荧光素酶报告基因实验检测circPVT1与miR-195-5p、miR-195-5p与SOX9的靶向关系。结果:与NCM-460细胞比较,SW480细胞、HCT116细胞、HCT29细胞、LOVO细胞、SW620细胞中circPVT1、SOX9 mRNA表达显著升高,miR-195-5p表达显著降低(P<0.05);其中,SW480细胞中circPVT1、SOX9 mRNA表达最高,miR-195-5p表达最低;故选择SW480细胞进行后续实验。与对照组和空载质粒组比较,circPVT1沉默组circPVT1、SOX9 mRNA及蛋白表达显著降低,miR-195-5p表达显著升高(P<0.05);miR-195-5p过表达组miR-195-5p表达显著升高,SOX9 mRNA及蛋白表达显著降低(P<0.05);SOX9沉默组SOX9 mRNA及蛋白表达显著降低(P<0.05)。与对照组和空载质粒组比较,circPVT1沉默组、miR-195-5p过表达组、SOX9沉默组细胞增殖率、Bcl-2、Vimentin蛋白表达显著降低,凋亡率、Bax、E-cadherin蛋白表达显著升高(P<0.05)。与circPVT1沉默组比较,circPVT1沉默+miR-195-5p低表达组miR-195-5p、凋亡率、Bax、E-cadherin蛋白表达显著降低,SOX9 mRNA及蛋白、细胞增殖率、Bcl-2、Vimentin蛋白表达显著升高(P<0.05)。circPVT1与miR-195-5p、miR-195-5p与SOX9存在靶向调控关系。结论:沉默circPVT1可以靶向上调miR-195-5p表达,抑制SOX9表达,进而抑制SW480细胞增殖和EMT进程,促进其凋亡。

Objective:To investigate the effects of circular RNA plasma cytoma variant translocation 1(circPVT1)on the proliferation,apoptosis and epithelial-mesenchymal transformation(EMT)of colon cancer cells by targeting microRNA-195-5p(miR-195-5p)/sex determining region Y box protein 9(SOX9)axis.Methods:In vitro culture of human normal colon epithelial cells NCM-460 and human colon cancer cell lines(SW480,HCT116,HCT29,LOVO,SW620)to logarithmic stage,SW480 cells were divided into control group(normal culture SW480 cells were not transfected),empty plasmid group(transfected with empty plasmid),circPVT1 silencing group[transfected with circPVT1 small interfering RNA(siRNA)plasmid],miR-195-5p overexpression group(transfected with miR-195-5p mimics plasmid),SOX9 silencing group(transfected with SOX9 siRNA plasmid),circPVT1 silencing+miR-195-5p low expression group(transfected with circPVT1 siRNA and miR-195-5p siRNA plasmid).Each group was transfected with corresponding plasmid and cultured for 48 h.The expression of circPVT1,miR-195-5p and SOX9 mRNA in NCM-460 cells,human colon cancer cell lines(SW480,HCT116,HCT29,LOVO,SW620)and SW480 cells in each group was detected by fluorescence quantitative PCR.The proliferation and apoptosis of SW480 cells in each group were detected by thiazole blue and flow cytometry.The expression of SOX9,apoptosis and EMT-related proteins in SW480 cells were detected by western blotting.The targeting relationship between circPVT1 and miR-195-5p,miR-195-5p and SOX9 was detected by dual luciferase reporter gene assay.Results:Compared with NCM-460 cells,the expressions of circPVT1 and SOX9 mRNA in SW480 cells,HCT116 cells,HCT29 cells,LOVO cells and SW620 cells were significantly increased,while the expression of miR-195-5p was significantly decreased(P<0.05).The expressions of circPVT1 and SOX9 mRNA were the highest in SW480 cells,and the expression of miR-195-5p was the lowest.Therefore,SW480 cells were selected for follow-up experiments.Compared with control group and empty plasmid group,the expressions of circPVT1,SOX9 mRNA and protein in circPVT1 silencing group were significantly decreased,while the expression of miR-195-5p was significantly increased(P<0.05).The expression of miR-195-5p in miR-195-5p overexpression group was significantly increased,while the expression of SOX9 mRNA and protein was significantly decreased(P<0.05).The expression of SOX9 mRNA and protein in SOX9 silencing group was significantly decreased(P<0.05).Compared with control group and empty plasmid group,the cell proliferation rate,the expression of Bcl-2 and Vimentin protein in circPVT1 silencing group,miR-195-5p overexpression group and SOX9 silencing group were significantly decreased,while the apoptosis rate,Bax and E-cadherin protein expressions were significantly increased(P<0.05).Compared with circPVT1 silencing group,miR-195-5p expression,apoptosis rate,Bax,E-cadherin expression in the circPVT1 silencing+miR-195-5p low expression group were significantly decreased,while the expression of SOX9 mRNA and protein,cell proliferation rate,Bcl-2,Vimentin expression were significantly increased(P<0.05).circPVT1 and miR-195-5p,miR-195-5p and SOX9 have targeted regulatory relationships.Conclusion:Silencing circPVT1 can up regulate the expression of miR-195-5p,inhibit the expression of SOX9,thereby inhibit the proliferation and EMT process of SW480 cells,and promote their apoptosis.