过表达miR-136-5p对子宫内膜癌HEC-1A细胞增殖、侵袭以及凋亡的影响

The effect of overexpression of miR-136-5p on the proliferation, invasion and apoptosis of endometrial carcinoma cells

目的探讨miR-136-5p和E2F1在子宫内膜癌中的表达以及过表达miR-136-5p对子宫内膜癌细胞增殖、侵袭和凋亡的影响。方法通过生物信息学网站分析TCGA数据库中子宫内膜癌miR-136-5p以及E2F1mRNA和蛋白的表达。采用miR-136-5p模拟物转染子宫内膜癌HEC-1A细胞,设置空白组(未进行转染)、miR-136-5p阴性对照组(转染无义序列)、miR-136-5p模拟物转染组(转染miR-136-5p模拟物),采用CCK-8方法检测各组HEC-1A细胞的增殖能力,Transwell实验检测各组HEC-1A细胞的侵袭能力,流式细胞仪检测细胞凋亡情况,Western blot检测HEC-1A细胞E2F1蛋白表达,应用Targetscan软件预测E2F1是否为miR-136-5p的靶基因,应用双荧光素酶报告基因实验验证。结果生物信息学分析显示,与正常组相比,子宫内膜癌组织中miR-136-5p表达下调,E2F1的mRNA和蛋白表达上升。miR-136-5p模拟物转染组HEC-1A细胞中miR-136-5p表达上升,细胞增殖能力下降、侵袭能力下降,细胞凋亡率上升。miR-136-5p过表达组HEC-1A细胞中E2F1蛋白表达下降,生物信息学分析结果显示,E2F1是miR-136-5p的靶基因,双荧光素酶实验证实E2F1为miR-136-5p的靶基因。结论过表达miR-136-5p可以抑制HEC-1A细胞增殖与侵袭,促进凋亡,其机制可能与调控E2F1的表达相关。

Objective To investigate the expression of miR-136-5 p and E2F1 in endometrial carcinoma and the effect and mechanism of over expression of miR-136-5 p on proliferation, invasion and apoptosis of HEC-1 A cells. Methods The expression of mir-136-5 p and E2F1 mRNA and protein in TCGA database was analyzed by bioinformatics website. miR-136-5 p mimics was transfected into HEC-1 A cells, and divided into three groups: blank group(not transfected), miR-136-5 p negative control group(transfected nonsense sequence), miR-136-5 p mimics group(transfected miR-136-5 p mimics). CCK-8 was used to detect the proliferation, transwell was used to detect the invasion, flow cytometry was used to detect the apoptosis, Western blot was used to detect the expression of E2F1 protein, and whether E2F1 was the target gene of mir-136-5 p was predicted by Targetscan 7.2, which was verified by double luciferase reporter gene experiment. Results Bioinformatics analysis showed that the expression of miR-136-5 p was down regulated and E2F1 was up regulated in endometrial carcinoma compared with normal group. After over-expression of miR-136-5 p in HEC-1 A cells, the expression of E2F1 was down-regulated, and the over-expression of miR-136-5 p could inhibit the proliferation and invasion, and promote the apoptosis of HEC-1 A cells. Bioinformatics analysis showed that E2F1 was one of the target genes of miR-136-5 p, and dual luciferase reporter gene confirmed that E2F1 was the target gene of miR-136-5 p. Conclusion The overexpression of miR-136-5 p may inhibit the proliferation and invasion, and promote the apoptosis in HEC-1 A cells, which may be related to the regulation of E2F1 expression.