微小RNA-148a-3p靶向调控MAP3K9表达及对胃癌细胞增殖和凋亡的影响

目的探讨微小RNA-148a-3p(miR-148a-3p)对丝裂原活化蛋白激酶激酶激酶9(MAP3K9)的靶向调控作用及对胃癌细胞增殖和凋亡的影响。方法向对数生长期胃癌细胞株MGC-803转染miR-148a-3p模拟物(mimics组)和阴性对照(NC组),以未转染的MGC-803细胞为对照组;采用实时定量PCR(QPCR)检测各组miR-148a-3p水平以评价转染效率,MTT法检测各组细胞增殖能力,流式细胞术检测各组细胞凋亡情况,分别采用QPCR和Western blotting检测Bcl-2、Bax、caspase-3及MAP3K9 mRNA和蛋白水平,同时采用双荧光素酶报告实验验证miR-148a-3p与MAP3K9的靶向作用关系。结果QPCR结果显示,对照组、NC组和mimics组的miR-148a-3p水平分别为1. 021±0. 123、1. 087±0. 196和2. 854±0. 368,与对照组和NC组比较,mimics组的miR-148a-3p水平升高(P<0. 05)。mimics组MGC-803细胞的增殖活力较其余两组减弱(P<0. 05)。mimics组MGC-803细胞凋亡率为(15. 2±1. 6)%,高于对照组的(3. 5±0. 9%)%和NC组的(4. 5±1. 1)%,差异具有统计学意义(P<0. 05)。与对照组和NC组比较,mimics组的MAP3K9和Bcl-2的mRNA和蛋白水平均下调,而Bax和caspase-3的mRNA和蛋白水平均上调(P<0. 05);双荧光素酶报告实验证实MAP3K9是miR-148a-3p的直接作用靶点。结论 MiR-148a-3p可抑制胃癌细胞MGC-803的增殖并诱导其凋亡,可能通过靶向MAP3K9来发挥抑癌作用,调控miR-148a-3p/MAP3K9轴在胃癌防治中有一定应用前景。

Targeted regulation of MAP3K9 expression by microRNA-148a-3p and its effect on proliferation and apoptosis of gastric cancer cells

Objective To investigate the targeted regulation of mitogen-activated protein kinase 9 （MAP3K9） expression by microRNA-148a-3p （miR-148a-3p） and its effect on proliferation and apoptosis of gastric cancer cells. MethodsGastric cancer MGC-803 cells at the logarithmic growth phase were transfected with miR-148a-3p mimics （mimics group） and negative control （NC group）， and the untransfected MGC-803 cells were used as control group. Real-time quantitative PCR （QPCR） was used to detect the expression of miR-148a-3p after transfection in each group to evaluate the transfection efficiency. MTT proliferation assay was conducted to evaluate the proliferation. Flow cytometry was used to detect apoptosis in each group. The expression of Bcl-2， Bax， caspase-3 and MAP3K9 were detected by QPCR and Western blotting， respectively. The targeting relationship between miR-148a-3p and MAP3K9 was verified by double luciferase report assay. Results The Results of QPCR showed that the expression of miR-148a-3p in control group， NC group and mimics group were 1.021±0.123， 1.087±0.196 and 2.854±0.368， respectively. Compared with control group and NC group， the expression of miR-148a-3p in mimics group was higher （P＜0.05）. The proliferation activity of MGC-803 cells in mimics group was weaker than that in the other two groups （P＜0.05）. The apoptotic rate in mimics group was （15.2±1.6）％， higher than （3.5±0.9）％ of control group and （4.5±1.1）％ of NC group （P＜0.05）. Compared with other two groups， the expressions of MAP3K9 and Bcl-2 were down-regulated in the mimics group， while the expressions of Bax and caspase-3 were up-regulated significantly （P＜0.05）. Double luciferase assay confirmed that MAP3K9 was the direct target of miR-148a-3p. Conclusion MiR-148a-3p can inhibit the proliferation and induce apoptosis of MGC-803 cells as anti-cancer factor possibly by targeting MAP3K9. MiR-148a-3p／MAP3K9 axis has a certain application prospect in the prevention and treatment of gastric cancer.