摘要目的 探讨微RNA-204(miR-204)对宫颈癌Hela细胞侵袭性的影响及其机制.方法 将Hela细胞分为4组,阴性对照组加入随机序列对照物,miR-204模拟物组和miR-204抑制物组分别加入miR-204模拟物(agomir-204)和miR-204抑制物(antagomir-204),空白对照组不进行处理.72 h后收集细胞,荧光定量实时PCR检测各组细胞miR-204水平,免疫印迹法检测各组细胞埃兹蛋白(EZR)表达.使用在线工具预测EZR基因3′非翻译区(3′UTR)序列中的miR-204靶向序列.将EZR基因上的miR-204靶向序列克隆到双荧光素酶报告载体上,载体分别与agomir-204(miR-204模拟物组)、antagomir-204(miR-204抑制物组)、随机序列对照物(阴性对照组)共转染Hela细胞,设空白对照组不进行转染,检测各组细胞双荧光素酶相对活性比值.Transwell法检测miR-204对Hela细胞侵袭性的影响.结果 荧光定量实时PCR显示,与空白对照组和阴性对照组比较,miR-204模拟物组Hela细胞miR-204表达显著增加,而miR-204抑制物组miR-204表达显著降低(均P<0.05).免疫印迹显示,随着miR-204表达增加EZR蛋白表达下调.预测软件发现EZR基因3′UTR序列上1个miR-204结合靶点,测序验证显示靶点序列已经正确克隆入双荧光素酶报告载体.双荧光素酶报告基因系统检测显示,与空白对照组和阴性对照组比较,miR-204模拟物组双荧光素酶相对活性比值显著下调,而miR-204抑制物组双荧光素酶相对活性比值显著上调(均P<0.05).Transwell法检测显示,与空白对照组和阴性对照组比较,miR-204模拟物组穿膜细胞数显著减少,而miR-204抑制物组穿膜细胞数显著增加.结论 miR-204能够通过作用于EZR基因的靶点序列负调控宫颈癌Hela细胞EZR蛋白表达,并抑制细胞的侵袭性.

abstractsObjective To investigate the effect of microRNA-204(miR-204)on the invasion of the cervical cancer Hela cells and its underlying mechanism. Methods Hela cells were assigned to 4 groups. The negative control group was added with random microRNA sequences. miR-204 mimetic(agomir-204) and miR-204 inhibitor(antagomir-204)were added to the miR-204 mimetic group and the miR-204 inhibitor group. A blank control group was not processed. At 72 h,the Hela cells were retrieved. Fluorescence quantitative real-time PCR was used to detect miR-204 levels in each group. The expression of Ezrin(EZR) protein was detected by immunoblotting. The miR-204 targeting sequence in the 3′-untranslated region(3′-UTR)of EZR gene was predicted using an online tool. The miR-204 targeting sequence was cloned into a dual luciferase reporter vector. With this vector,the Hela cells were co-transfected with agomir-204(miR-204 mimetic group),antagomir-204(miR-204 inhibitor group)and random miRNA sequence(negative control group),respectively. The blank control group was not transfected. The relative luciferase activity in each group was detected. Transwell assay was used to detect the effect of miR-204 on the invasion of Hela cells. Results Fluorescence quantitative real-time PCR showed that miR-204 expression in the miR-204 mimetic group of Hela cells was significantly increased and significantly decreased in the miR-204 inhibitor group,compared with the blank control group and negative control group (all P<0.05). Immunoblotting showed a decrease in EZR protein along with the increasing miR-204 expression. The prediction software found a miR-204 binding target on the 3′-UTR of EZR gene. By gene sequencing,the target sequence was confirmed to have been correctly cloned into the dual luciferase reporter vector. Compared with the blank control group and negative control group,the relative luciferase activity was down-regulated in the miR-204 mimetic group and up-regulated in the miR-204 inhibitor group (all P<0.05). Transwell assay showed a significant reduction in the number of transmembrane cells in the miR-204 mimetic group compared to the blank control group and the negative control group,whereas the number of transmembrane cells in the miR-204 inhibitor group increased significantly. Conclusion miR-204 can down-regulate the EZR protein through the target sequence of EZR gene in cervical cancer Hela cells,and inhibit the invasion of the cells.