miR-103通过NF-κB通路上调MGMT表达诱导胃癌细胞紫杉醇耐药

miR-103 induces paclitaxel resistance in gastric cancer cells through NF-κB pathway-upregulated MGMT expression

目的观察miR-103在胃癌细胞紫杉醇(PTX)耐药中的作用,并探讨其作用机制。方法构建PTX耐药胃癌NCI-N87细胞(N87/PTX);N87/PTX细胞转染miR-103 inhibitor和阴性对照,分别设为miR-103 inhibitor组和阴性对照组(NC),同时以未转染的N87/PTX细胞作为空白对照组(Control)。四甲基偶氮唑盐比色法(MTT)检测细胞活力;实时荧光定量PCR(RT-qPCR)检测细胞miR-103表达水平;生物信息学分析miR-103的靶基因;结晶紫染色法检测细胞贴壁存活率;免疫荧光法检测细胞NF-κB p65定位;蛋白印记法检测p-NF-κB p65、IKBα、MGMT蛋白表达变化。结果N87/PTX细胞耐药指数为27.1。与正常N87细胞比较,N87/PTX细胞miR-103表达水平显著升高(P<0.01)。miR-103 inhibitor可显著降低PTX对N87/PTX细胞的IC50(P<0.01),增强N87/PTX细胞对PTX的敏感性(P<0.01)。IKBα蛋白的编码基因NFKBIA与miR-103存在互补结合位点。与正常N87细胞比较,N87/PTX细胞IKBα表达明显下调,p-NF-κBp65和MGMT表达显著升高(P均<0.01)。抑制miR-103可上调IKBα蛋白表达,下调p-NF-κB p65和MGMT蛋白表达(P均<0.01)。结论miR-103可能通过降解IKBα诱导NF-κB p65入核,NF-κB p65增强MGMT转录表达从而介导胃癌细胞对PTX耐药。

Objective To observe the role of miR-103 in gastric cancer cell resistance to paclitaxel(PTX)and then explore the underlying molecular events.Methods Gastric cancer NCI-N87 cells were used to establish PTX-resistant cell subline(N87/PTX)and then transfected with miR-103 inhibitor and negative control as miR-103 inhibitor and negative control(NC)groups,respectively.The untransfected N87/PTX cells were used as a blank control(Control).Cell viability was detected by tetramethylazolium salt colorimetry(MTT)assay.The expression of miR-103 in cells was detected using real-time quantitative PCR(RT-qPCR).Bioinformatical analysis was performed to analyze the miR-103-targeting genes.Crystal violet staining was used to detect cell adherent viability.The localization of NF-κB p65 in cells was detected by immunofluorescence.Level of p-NF-κB p65,IKBα,and MGMT was detected by Western blot.Results The drug resistance index of N87/PTX cells was 27.1.Compared with parental N87 cells,miR-103 level was significantly upregulated in N87/PTX cells(P<0.01).Moreover,the miR-103 inhibitor significantly reduced the IC50 of PTX in N87/PTX cells(P<0.01)and enhanced the sensitivity of N87/PTX cells to PTX treatment(P<0.01).In addition,the gene encoding IKBαprotein,NFKBIA,contained complementary binding sites to miR-103.Thus,compared to parental N87 cells,expression of IKBαin N87/PTX cells was significantly downregulated and expression of p-NF-κBp65 and MGMT was significantly upregulated(all P<0.01).Inhibition of miR-103 expression was able to upregulate expression of IKBαprotein but downregulate level of p-NF-κB p65 and MGMT proteins(all P<0.01).Conclusion miR-103 was able to induce NF-κB p65 into the cell nucleus by degrading IKBα,while NF-κB p65 enhanced the transcriptional MGMT expression to mediate the resistance of gastric cancer cells to PTX.