微小RNA-494通过抑制第10号染色体上缺失与张力蛋白同源的磷酸酯酶基因的表达促进乳腺癌MCF-7细胞的增殖和转移

MicroRNA-494 promotes cell proliferation and migration in breast cancer cell MCF-7 by directly targeting phosphatase and tensin homologue deleted on chromosometen

目的 探讨微小RNA（miRNA,miR）-494对乳腺癌细胞MCF-7的增殖和转移的调控作用以及机制.方法 在乳腺癌细胞株MCF-7转染miR-494模拟物及抑制剂、阴性对照,然后采用噻唑蓝（MTT）方法检测miR494对MCF-7细胞活力的影响,并通过Transwell转移实验检测miR-494对MCF-7转移能力的影响.通过双荧光素酶实验检测miR-494的靶基因,并且用反转录-聚合酶链反应（RT-PCR）和Western blot进行验证.结果 转染miR-494 mimics后,细胞活力明显提高,24 h细胞活力比值为1.28,48 h后为1.80,（P＜0.05）,转染miR-494 inhibitor后,细胞活力显著下调,24h细胞活力比值为0.80,48 h后为0.68（P ＜0.05）.对照组细胞均数为67个/视野,转染miR-494mimics后的MCF-7细胞的均数为：122个/视野（P＜0.05）,转染miR-494 inhibitor后的MCF-7细胞的均数为：47个/视野（P＜0.01）.野生型载体模拟物组相对荧光素酶活性：0.69（P＜0.01）,抑制剂组：1.90（P＜0.01）;突变型载体模拟物组相对荧光素酶活性：1.09,抑制剂组：1.19.miR-494过表达后PTEN的mRNA和蛋白水平确实出现明显下降,相反地,miR-494的抑制却导致PTEN蛋白水平和mRNA水平的上调.结论 高表达miR-494可显著促进乳腺癌细胞株MCF-7的增殖与转移能力,抑制miR-494可显著降低MCF-7的增殖与转移能力,并且证实miR-494是通过靶向第10号染色体上缺失与张力蛋白同源的磷酸酯酶基因（PTEN）的3＇端非编码区域（3＇-UTR）来调节乳腺癌细胞株MCF-7的增殖和转移能力.

Objective To investigate whether microRNA （miR）-494 mediated cell proliferation and migration and the regulatory mechanism in breast cancer cell MCF-7.Methods miR-494 mimics and inhibitor and negative control （NC） were transfected into MCF-7 cells, and cell viability was evaluated by methyl thiazol tetrazolium （MTT） assay.Cell migrasion was assessed by Transwell migrasion assay.Dual-luciferase reporter assay, reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting were used to identify the target gene of miR-494.Results After the transfection of miR-494 mimics, the cell vitality were improved obviously, cell vitality ratio after 24 h was ： 1.28, after 48h was ： 1.80.After the transfection of miR-494 inhibitor, the cell vitality were reduced obviously, cell vitality ratio after 24 h was： 0.80, after 48 h was ： 0.68.（all P 〈 0.05）.The mean of the control group is ： 67/view.The mimics group was 122/view （P 〈 0.05） , the inhibitor group was 47/view （P 〈 0.01）.Wild type cartier relative luciferase activity ： analog group ： 0.69 （P 〈 0.01）, the inhibitor groups ： 1.90 （P 〈 0.01）.Mutant carrier analog group relative luciferase activity： 1.09, inhibitor groups： 1.19.MiR-494 after expression of PTEN mRNA and protein levels significantly decline, in contrast, inhibition of miR-494 is lead to increase the level of PTEN protein and mRNA level.Conclusion These results suggested that miR-494 can regulate cell proliferation and migration through targeting PTEN in MCF-7 cells.