miR-496靶向调控CDK1促进肺癌A549细胞凋亡

The Effect of mi R-496 on Cell Apoptosis by Targeting CDK1 in Lung Cancer A549 Cells

为探讨miR-496对肺癌A549细胞的增殖和凋亡调控作用及分子机制,本研究通过荧光定量PCR检测miR-496在肺癌和癌旁组织中的表达,记A549细胞转染miR-496 mimics及对照miR-NC;通过CCK8检测miR-496对肺癌A549细胞增殖的影响,采用流式细胞术检测miR-496对A549细胞凋亡的影响,并通过双荧光素酶报告基因检测miR-496的靶基因;采用Western blotting检测miR-496对靶基因CDK1蛋白表达的影响。荧光定量PCR结果显示,肺癌组中的miR-496的相对表达量较癌旁组比较表达显著下调(p<0.01)。CCK8结果显示,miR-496 mimics组细胞在培养24 h和48 h后细胞增值率(A450值)较miR-NC对照组显著降低。流式细胞术检测结果显示,miR-496 mimics组细胞凋亡率较miR-NC对照组显著增加。双荧光素酶报告基因实验结果显示,miR-496 mimics与CDK13’UTR WT载体共转染组荧光强度明显低于对照miR-NC与WT载体共转染组。Western blotting检测结果显示,miR-496 mimics组细胞的CDK1蛋白表达量与miR-NC对照组比较显著降低(p<0.01),miR-496 mimics+CDK1组细胞CDK1蛋白表达量与miR-496mimics组比较显著升高(p<0.01)。miR-496能够通过抑制靶基因CDK1的表达,抑制肺癌细胞A549的增殖,促进肺癌A549细胞凋亡。本研究结果有助于了解miR-496和CDK1在肺癌中的作用,为肺癌的治疗提供理论依据和实验基础。

To investigate the role and molecular mechanism of miR-496 on proliferation and apoptosis of A549 cells in lung cancer.The expression of miR-496 in lung cancer and paracarcinoma tissues was detected by Q-PCR in this reseach.The downstream target gene of miR-496 was detected by dual luciferase reporter gene assay.A549 cells were transferred with miR-NC and miR-496 mimics.The cell viability was detected by CCK8 assay.The apoptosis of A549 cells was detected by flow cytometer.The expression of CDK1 protein was detected by Western blotting.The results of Q-PCR showed that the relative expression of miR-496 in the lung cancer tissues was significantly lower than that in the paracarcinoma tissues(p<0.01).CCK8 results showed that the cell proliferation rate(A450)of miR-496 mimics group after transfection at 24 h and 48 h were significantly lower,compared with miR-NC control group.Flow cytometry results showed that the apoptosis of A549 cells in miR-496 mimics group was significant higher than that in the miR-NC group.The dual luciferase reporter assay results showed that the fluorescence intensity of miR-496 mimics co-transfected with CDK13’UTR WT vector was significantly lower than that of the control miR-NC and WT vector co-transfection group.Western blotting results showed that the expression of CDK1 protein in the miR-496 mimics group was significantly lower than that in the miR-NC control group(p<0.01).The expression of CDK1 protein in the miR-496 mimics+CDK1 group was significantly higher than that in the miR-496 mimics group(p<0.01).miR-496 inhibited the proliferation of lung cancer A549 cells and promoted apoptosis by regulating the expression of target gene CDK1.The results of this study are helpful to understand the role of miR-496 and CDK1 in lung cancer,and provide theoretical basis and experimental basis for the treatment of lung cancer.