miR-379-5p靶向CTSL调控肺癌细胞增殖、迁移、侵袭及凋亡的分子机制

Molecular mechanism of miR-379-5p regulating proliferation,migration,invasion and apoptosis of lung cancer cells by targeting CTSL

目的:探讨微小RNA-379-5p(miR-379-5p)通过靶向组织蛋白酶L(CTSL)调控肺癌细胞增殖、迁移、侵袭及凋亡的作用机制。方法:选择本院收治的肺癌患者57例,取患者肺癌组织与癌旁组织,应用qRT-PCR法检测miR-379-5p与CTSL mRNA的表达水平;免疫组化法检测CTSL蛋白的表达情况。miR-con(miR-con组)、miR-379-5p mimics(miR-379-5p组)分别转染肺癌NC-H446细胞,miR-379-5p mimics与pcDNA(miR-379-5p+pcDNA组)、miR-379-5p mimics与pcDNA-CTSL(miR-379-5p+pcDNA-CTSL组)分别共转染肺癌NC-H446细胞,MTT检测细胞增殖情况;流式细胞术检测细胞凋亡率;Transwell迁移与侵袭实验检测细胞迁移与侵袭能力。双荧光素酶报告实验验证miR-379-5p与CTSL之间的靶向关系。Western blot检测CTSL、Cyclin D1、MMP-2、MMP-9、Cleaved caspase-3蛋白的表达。结果:肺癌组织中miR-379-5p的表达水平显著低于癌旁组织(P<0.05),而CTSL mRNA及蛋白阳性率均显著高于癌旁组织(P<0.05);与miR-con组比较,miR-379-5p组细胞增殖、迁移、侵袭能力显著降低(P<0.05),细胞凋亡率显著增加(P<0.05),明显抑制Cyclin D1、MMP-2、MMP-9蛋白表达(P<0.05),而促进Cleaved caspase-3蛋白表达(P<0.05);双荧光素酶报告实验证明miR-379-5p可负向调控CTSL表达与活性;CTSL过表达可逆转miR-379-5p过表达对肺癌细胞增殖、迁移、侵袭及凋亡的调控作用。结论:miR-379-5p过表达通过靶向CTSL而减弱肺癌细胞增殖、迁移及侵袭能力,诱导细胞凋亡。

Objective:To investigate the mechanism of microRNA-379-5p(miR-379-5p)regulating the proliferation,migration,invasion and apoptosis of lung cancer cells by targeting cathepsin L(CTSL).Methods:A total of 57 patients with lung cancer admitted to our hospital were enrolled.The lung cancer tissues and adjacent tissues were taken.The expression levels of miR-379-5p and CTSL mRNA were detected by qRT-PCR.The expression of CTSL protein was detected by immunohistochemical method.miR-con(miR-con group),miR-379-5p mimics(miR-379-5p group)were transfected into lung cancer NC-H446 cells.miR-379-5p mimics and pcDNA(miR-379-5p+pcDNA group),miR-379-5p mimics and pcDNA-CTSL(miR-379-5p+pcDNA-CTSL group)were co-transfected into lung cancer NC-H446 cells,respectively.MTT was used to detect cell proliferation.Apoptosis rate was measured by flow cytometry.Transwell migration and invasion assays were used to detect cell migration and invasion.The dual luciferase reporter assay validated the targeting relationship between miR-379-5p and CTSL.The expressions of CTSL,Cyclin D1,MMP-2,MMP-9 and Cleaved caspase-3 were detected by Western blot.Results:The expression level of miR-379-5p in lung cancer tissues was significantly lower than that in adjacent tissues(P<0.05),and the positive rates of CTSL mRNA and protein were significantly higher than those in adjacent tissues(P<0.05).Compared with the miR-con group,the proliferation,migration and invasion ability of the miR-379-5p group were significantly decreased(P<0.05),and the apoptosis rate was significantly increased(P<0.05).The expression of Cyclin D1,MMP-2 and MMP-9 protein was significantly inhibited(P<0.05),while the expression of Cleaved caspase-3 protein was significantly promoted(P<0.05).Dual luciferase reporter assay demonstrated that miR-379-5p could negatively regulate CTSL expression and activity.Overexpression of CTSL reversed the regulation of miR-379-5p overexpression on proliferation,migration,invasion and apoptosis of lung cancer cells.Conclusion:Overexpression of miR-379-5p attenuates the proliferation,migration and invasion of lung cancer cells by targeting CTSL,and induces apoptosis.