miR-145抑制口腔鳞状细胞癌Tca-8113细胞增殖活性及其与CDK6的靶向关系  

Inhibitive effect of miR-145 on the proliferation of oral squamous cell carcinoma cell line Tca-8113 and its relationship with the targeting CDK6

作　　者：郭芳[1] 黄硕[1] 

机构地区：[1]西安医学院口腔医学系,西安710021

出　　处：《山西医科大学学报》2014年第7期576-580,共5页Journal of Shanxi Medical University

基　　金：陕西省教育厅自然科学专项基金资助项目(2013JK0790)

摘　　要：目的在口腔鳞状细胞癌Tca-8113细胞中验证miR-145靶向CDK6对其细胞增殖活性的影响。方法构建pcDNATM6.2-GW-pre-miR-145重组表达质粒并转染入Tca-8113细胞,以荧光实时定量PCR(qRT-PCR)检测转染后癌细胞miR-145的表达水平。MTT法评价miR-145对Tca-8113细胞增殖活性的影响,并通过双荧光素酶报告基因检测miR-145对候选靶基因CDK6的调控作用,同时采用Western blot法检测miR-145对CDK6蛋白表达水平的影响。结果成功构建miR-145真核表达载体,qRT-PCR显示miR-145组中miR-145的表达显著高于内源性表达量(P<0.001),说明miR-145在Tca-8113细胞中具有较高的转染效率。MTT法提示miR-145的过表达能够显著抑制Tca-8113细胞的增殖(P<0.01),同时双荧光素酶报告基因分析表明,共同转入miR-145与CDK6野生型(CDK6-WT)会显著抑制Tca-8113细胞中的荧光酶表达(P=0.002);Western blot进一步证实了miR-145的过表达能够抑制内源性CDK6蛋白的表达。结论 miR-145可能通过直接靶向CDK6来抑制口腔鳞状细胞癌Tca-8113细胞的增殖活性。Objective To explore the effects of miR-145 targeting CDK6 on Tca-8113 cell lines cell proliferation activity.Methods The pcDNATM6.2-GW-pre-miR-145 recombinant expression plasmid was constructed and then transfected into Tca-8113 cells.And then,the expression level of miR-145 was detected after transfection using quantitative real-time PCR （qRT-PCR）.Effect of miR-145 on Tca-8113 cell proliferation activity was evaluated by MTT assay,and the regulation of miR-145 on the target gene CDK6 was detected by dual luciferase reporter gene,and the effect of miR-145 on CDK6 expression levels was detected by Western blot.Results miR-145 eukaryotic expression vector was successfully constructed.The qRT-PCR results showed that miR-145 expression was significantly higher than the expression of the endogenous expression levels after transfection of pcDNATM6.2-GW-pre-miR-145 recombinant expression plasmid （P ＜ 0.001）,which indicated that miR-145 had a high transfection efficiency in Tca-8113 cells.MTT assay results showed that overexpression of miR-145 significantly inhibited the proliferation of Tca-8113 cells（P ＜ 0.01）.Meanwhile,the gene analysis of dual luciferase reporter showed that the fluorescence activity was significantly inhibited after cotransfection of miR-145 and CDK6 wildtype （CDK6-WT） in Tca-8113 cells （P =0.002）.Western blot result further confirmed that overexpression of miR-145 inhibited the expression of endogenous CDK6.Conclusion miR-145 may inhibit oral squamous cell carcinoma Tca-8113 cell proliferation by directly targeting CDK6.

关 键 词：口腔鳞状细胞癌 MIR-145 CDK6 

分 类 号：R739.8[医药卫生—肿瘤]