miR-1-3p通过靶向调控CDK9对非小细胞肺癌细胞增殖和凋亡的影响  

Effect of miR-1-3p on proliferation and apoptosis of non-small cell lung cancer cells by targeting CDK9

作　　者：黄尚校 李春君[2] 黄剑锋[1] 黄昌杰[3] HUANG Shang-Xiao;LI Chun-Jun;HUANG Jian-Feng;HUANG Chang-Jie(Department of Radiotherapy,the Second Nanning People’s Hospital,Nanning 530031,China)

机构地区：[1]南宁市第二人民医院放疗科,南宁530031 [2]广西医科大学附属肿瘤医院病理科,南宁530021 [3]南宁市第二人民医院肿瘤内科,南宁530031

出　　处：《中国免疫学杂志》2020年第20期2468-2472,2478,共6页Chinese Journal of Immunology

基　　金：广西省自然科学基金项目(No.2010GXNSFA013260)。

摘　　要：目的:探讨miR-1-3p与细胞周期蛋白依赖性激酶9(CDK9)的靶向调控关系及其对非小细胞肺癌(NSCLC)细胞增殖和凋亡的影响。方法:培养人肺上皮细胞株BEAS-2B及人NSCLC细胞株A549、H1299、HCC827和H460,并将miR-1-3p mimic及其阴性对照(miR-NC)转染至A549细胞,qPCR法检测细胞miR-1-3p表达水平,Western blot法检测细胞CDK9蛋白表达水平,生物信息学方法预测miR-1-3p可能的靶基因,双荧光素酶报告基因实验验证miR-1-3p与CDK9的靶向调控关系。将miR-1-3p mimic和pCEP4-CDK9质粒分别或同时转染至A549细胞,CCK-8法检测细胞增殖率,平板克隆形成实验检测细胞克隆形成能力,流式细胞术检测细胞凋亡率,Western blot法检测凋亡相关蛋白cleaved-Caspase-3、Bax和Bcl-2蛋白表达水平。结果:与BEAS-2B细胞相比,A549、H1299、HCC827和H460细胞中miR-1-3p表达降低(P<0.05),CDK9蛋白表达升高(P<0.05),A549细胞水平变化最为显著。CDK9是miR-1-3p的靶基因,可被miR-1-3p靶向负调控抑制其蛋白表达。miR-1-3p过表达可显著抑制A549细胞增殖及细胞克隆形成能力,提高A549细胞凋亡率,促进cleaved-Caspase-3和Bax蛋白表达,抑制Bcl-2蛋白表达。过表达CDK9可明显逆转miR-1-3p对A549细胞增殖的抑制作用及凋亡的促进作用。结论:miR-1-3p可抑制NSCLC细胞增殖并促进其凋亡,其机制可能与靶向抑制CDK9蛋白表达有关。Objective:To investigate targeted regulatory relationship between miR-1-3p and CDK9,and its effects on proliferation and apoptosis of non-small cell lung cancer(NSCLC)cells.Methods:Human lung epithelial cell line BEAS-2B and human NSCLC cell lines A549,H1299,HCC827 and H460 were cultured,and miR-1-3p mimic and its negative control(miR-NC)were transfected into A549 cells.Expression level of miR-1-3p was detected by qPCR and protein expression level of CDK9 was detected by Western blot.Bioinformatics analysis was used to predict potential target of miR-1-3p,and targeting effect of miR-1-3p on CDK9 was verified by dual-luciferase reporter assay system.miR-1-3p mimic and pCEP4-CDK9 plasmids were transfected into A549 cells separately or simultaneously,and cell proliferation rate was detected by CCK-8 method,ability of cell clone formation was detected by plate colony formation assay,apoptosis rate of A549 cells was detected by flow cytometry,and expression levels of apoptosis-related proteins cleaved-Caspase-3,Bax and Bcl-2 were detected by Western blot.Results:Compared with BEAS-2B cells,miR-1-3p expressions in A549,H1299,HCC827 and H460 cells were significantly decreased(P<0.05),while expression levels of CDK9 proteins were significantly increased(P<0.05),and level changes in A549 cells were the most significant.CDK9 is a target gene of miR-1-3p,which can be negatively regulated by miR-1-3p to inhibit its protein expression.Overexpression of miR-1-3p can significantly inhibit proliferation and ability of cell clone formation of A549 cells,increase apoptosis rate,promote protein expressions of cleaved-Caspase-3 and Bax,and inhibit protein expression of Bcl-2.Overexpression of CDK9 can significantly reverse effect of miR-1-3p on inhibitory of proliferation and promotion of apoptosis of A549 cells.Conclusion:miR-1-3p can inhibit proliferation and promote apoptosis of NSCLC lung cancer cells,whose mechanism may be related to targeted inhibition of CDK9 protein expression.

关 键 词：miR-1-3p 非小细胞肺癌 细胞周期蛋白依赖性激酶9 增殖 凋亡 

分 类 号：R734.2[医药卫生—肿瘤]