摘要
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。
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.Methods Gastric 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.
作者
牛虹
田同德
唐静雯
岳光星
李华华
范伊晓
周浩本
NIU Hong;TIAN Tongde;TANG Jingwen;YUE Guangxing;LI Huahua;FAN Yixiao;ZHOU Haoben(Department of Oncology,Integrative Medicine,Cancer Hospital Affiliated to Zhengzhou University,Zhengzhou 450008,China)
出处
《临床肿瘤学杂志》
CAS
北大核心
2019年第2期108-112,共5页
Chinese Clinical Oncology
