沉默TRPM2-AS对耐奥沙利铂的结直肠癌细胞增殖和凋亡的影响及机制研究

Effect of silencing TRPM2-AS on proliferation and apoptosis of oxaliplatin-resistant colorectal cancer cells and its mechanism

目的探讨长链非编码RNA(lncRNA)瞬时受体电位通道M2反义RNA(TRPM2-AS)对耐奥沙利铂(L-OHP)的结直肠癌细胞增殖和凋亡的影响及可能机制。方法采用L-OHP浓度递增处理结直肠癌细胞HCT-8,获得L-OHP耐药细胞HCT-8/L-OHP,RT-qPCR法分别检测HCT-8和HCT-8/L-OHP细胞中TRPM2-AS与miR-22-3p表达。分别转染TRPM2-AS小干扰RNA、miR-22-3p模拟物,或共转染TRPM2-AS小干扰RNA与miR-22-3p抑制剂至HCT-8/L-OHP细胞,CCK-8法和克隆形成实验检测细胞增殖,流式细胞术检测细胞凋亡,Western blot检测细胞cleaved-caspase3蛋白表达。双荧光素酶报告基因实验验证TRPM2-AS和miR-22-3p调控关系。结果HCT-8/L-OHP细胞中TRPM2-AS表达明显高于HCT-8细胞(P<0.05),而miR-22-3p表达明显低于HCT-8细胞(P<0.05)。沉默TRPM2-AS或过表达miR-22-3p后,HCT-8/L-OHP细胞OD_(48 h/72 h)值和克隆形成数降低(P<0.05),而细胞凋亡率及cleaved-caspase3蛋白表达升高(P<0.05)。TRPM2-AS可靶向结合miR-22-3p,且沉默TRPM2-AS促进HCT-8/L-OHP细胞中miR-22-3p表达。抑制miR-22-3p逆转了沉默TRPM2-AS对HCT-8/L-OHP细胞增殖和凋亡的影响。结论沉默TRPM2-AS可能通过靶向上调miR-22-3p阻碍耐L-OHP的结直肠癌细胞增殖,并促进其凋亡,TRPM2-AS可能是逆转结直肠癌细胞耐L-OHP的分子靶点。

Objective To investigate the effect of long chain non-coding RNA(lncRNA)TRPM2-AS on the proliferation and apoptosis of oxaliplatin(L-OHP)-resistant colorectal cancer cells and its possible mechanism.Methods Colorectal cancer cells HCT-8 were treated with increasing concentrations of L-OHP to obtain L-OHP resistant cells HCT-8/L-OHP.RT-qPCR was used to detect the expression of TRPM2-AS and miR-22-3p in HCT-8 and HCT-8/L-OHP cells.TRPM2-AS small interfering RNA or miR-22-3p mimics was transfected into HCT-8/L-OHP cells,or TRPM2-AS small interfering RNA and miR-22-3p inhibitors were co-transfected into HCT-8/L-OHP cells,and then CCK-8 method and clone formation experiment were used to detect cell proliferation,the flow cytometry was used to detect cell apoptosis,and Western blot was used to detect the expression of cleaved-caspase3 protein in cells.The dual luciferase reporter gene experiment verified the regulatory relationship between TRPM2-AS and miR-22-3p.Results The expression of TRPM2-AS in HCT-8/L-OHP cells was significantly higher than that in HCT-8 cells(P<0.05),but the expression of miR-22-3p was significantly lower than that in HCT-8 cells(P<0.05).After silencing TRPM2-AS or overexpressing miR-22-3p,the OD_(48 h/72 h) value and the number of clone formation of HCT-8/L-OHP cells were decreased(P<0.05),while the cellular apoptosis rate and the expression of cleaved-caspase3 protein were increased(P<0.05).TRPM2-AS could target to combined with miR-22-3p,moreover silencing TRPM2-AS promoted the expression of miR-22-3p in HCT-8/L-OHP cells.Inhibiting miR-22-3p reversed the effect of silencing TRPM2-AS on the proliferation and apoptosis of HCT-8/L-OHP cells.Conclusion Silencing TRPM2-AS may block the proliferation of L-OHP-resistant colorectal cancer cells and promote their apoptosis by targeting up-regulation of miR-22-3p.TRPM2-AS may be a molecular target spot to reverse the L-OHP resistance of colorectal cancer cells.