miR-126-5p靶向抑制ERCC1增加结肠癌细胞对奥沙利铂化疗的敏感性

miR-126-5p inhibits ERCC1 to increase sensitivity of colon cancer cells to oxaliplatin chemotherapy

目的探讨miR-126-5p通过靶向抑制切除修复交叉互补基因1(excision repair cross complement 1,ERCC1)促进结肠癌细胞对奥沙利铂(oxaliplatin,L-OHP)的敏感性及其分子机制。方法利用L-OHP浓度梯度递增法处理结肠癌细胞HCT116,建立耐L-OHP的人结肠癌细胞株HCT116/L-OHP。CCK-8法检测并计算L-OHP作用后的半数抑制浓度(half maximal inhibitory concentration,IC)值。qRT-PCR法检测miR-126-5p和ERCC1在HCT116和HCT116/L-OHP细胞株中的表达。利用Lipofectamine~?2000转染miR-126-5p mimics(miR-126-5p mimics组)和miR-NC质粒(miR-NC组)后,CCK-8法检测HCT116/L-OHP细胞对L-OHP的敏感性的变化,流式细胞术检测细胞凋亡率。利用双荧光素酶报告实验验证miR-126-5p和ERCC1的相互作用机制。Western blot法观察miR-126-5p mimics转染后ERCC1蛋白的表达变化。结果与HCT116细胞比较,HCT116/L-OHP细胞miR-126-5p表达水平下调(P<0.05)。不同浓度L-OHP(0、50、100、150和200μg/mL)处理48 h后,miR-126-5p mimics组细胞IC值低于miR-NC组和HCT116/L-OHP未转染组,miR-126-5p mimics的转染降低了HCT116/L-OHP细胞的增殖活性(均P<0.05)。转染48 h后,miR-126-5p mimics组较miR-NC组ERCC1蛋白表达下降(P<0.05)。双荧光素酶靶标实验表明,ERCC1是miR-126-5p的直接作用靶点。结论上调miR-126-5p表达可逆转HCT116/L-OHP细胞株对L-OHP的耐药性,其作用机制可能与负性调控ERCC1表达有关。

Objective To investigate the role of miR-126-5p in inhibiting excision repair cross complement 1(ERCC1)to increase the sensitivity to oxaliplatin(L-OHP)chemotherapy of colon cancer cells and its molecular mechanism.Methods A human colon carcinoma L-OHP resistant cell line HCT116/L-OHP was established in vitro from L-OHP treated human colon carcinoma HCT116 cells by the concentration gradient increasing method.CCK-8 was used to detect the half maximal inhibitory concentration(IC)of HCT116 and HCT116/L-OHP cells after L-OHP treatment.The expressions of miR-126-5p and ERCC1 in different cell lines were detected by qRT-PCR.HCT116/L-OHP cells were transfected with miR-126-5p mimics(miR-126-5p mimics group)and miR-NC plasmids(miR-NC group)by Lipofectamine2000.The effect of miR-126-5p on the sensitivity to L-OHP of the cells was detected by CCK-8 assay.The apoptosis rate was detected by flow cytometry.The interaction mechanism between miR-126-5p and ERCC1 was verified by dual luciferase assay.The protein expression of ERCC1 was detected by Western blot.Results The expression level of miR-126-5p in HCT116/L-OHP cells was down-regulated compared with HCT116 cells(P<0.05).After treatment with different concentrations of L-OHP(0,50,100,150,and 200μg/mL)for 48 h,the miR-126-5p mimics group had lower ICthan the miR-NC and untransfected HCT116/L-OHP groups,and miR-126-5p mimics reduced the proliferation of HCT116/L-OHP cells(all P<0.05).ERCC1 protein expression was significantly lower in the miR-126-5p mimics group than that in the miR-NC group at 48 h after transfection(P<0.05).Dual luciferase assay showed that ERCC1 was the direct target of miR-126-5p.Conclusions Upregulation of miR-126-5p expression can reverse the resistance of HCT116/L-OHP cells to L-OHP,and its mechanism may be related to the negative regulation of ERCC1 expression.