miR-146b-5p靶向调控NAIF1抑制胃癌细胞凋亡

MiR-146b-5p inhibits cells apoptosis by targeting NAIF1 in gastric cancer

目的分析miR-146b-5p对胃癌细胞MGC803增殖与凋亡的调控作用及分子机制。方法实时荧光定量PCR(qRT-PCR)检测不同胃癌细胞系(MGC803、BGC823、MKN28、MKN45和SGC7901)中miR-146b-5p水平;应用miR-146b-5p抑制剂(inhibitor)和阴性对照(miRNA-NC)分别转染胃癌细胞MGC803,MTT法检测细胞活力,流式细胞术检测细胞凋亡比例;TargetScan数据库分析miR-146b-5p的潜在靶基因,蛋白免疫印迹技术(western blot)检测核诱导凋亡因子1(NAIF1)蛋白表达水平;双荧光素酶实验验证miR-146b-5p和NAIF1之间的关系。结果不同胃癌细胞系(MGC803、BGC823、SGC7901、MKN45和MKN28)miR-146b-5p水平均高于正常人胃黏膜细胞(GES-1)(t分别=26.21、13.08、11.09、11.09、6.41,P均<0.05)。miR-146b-5p inhibitor转染胃癌细胞MGC803后miR-146b-5p水平明显降低(t=9.14,P<0.05)。抑制MGC803胃癌细胞miR-146b-5p水平后,细胞活力明显降低,细胞凋亡比例升高(t分别=3.73、9.55,P均<0.05)。miR-146b-5p mimics转染组WT-NAIF1荧光素酶活性明显低于miRNA-NC组(t=8.35,P<0.05)。Western blot结果表明,上调miR-146b-5p水平后,NAIF1蛋白表达明显降低(t=5.67,P<0.05),而下调miR-146b-5p水平后,NAIF1蛋白表达明显升高(t=4.32,P<0.05),在过表达NAIF1的基础上转染miR-146b-5p,细胞活力明显升高,细胞凋亡比例降低(t分别=6.54、4.73,P均<0.05)。结论miR-146b-5p通过靶向抑制NAIF1抑制胃癌细胞凋亡。

Objective To investigate the regulatory effect and molecular mechanism of miR-146b-5p on the proliferation and apoptosis of gastric cancer cell MGC803.Methods The expressions of miR-146b-5p in different gastric cancer cells were measured by reverse transcription-quantitative polymerase chain reaction(qRT-PCR).MGC803 cells were transfected with miR-146b-5p inhibitor or miRNA-NC respectively.The proliferation of MGC803 cells was measured by MTT,and the apoptosis of MGC803 cells was measured by flow cytometry.The potential targeting genes of miR-146b-5p were analyzed through TargetScan datebase.The expression of NAIF1 was measured by western blot.The relationship between miR-146b-5p and NAIF1 was discovered.Results The expression levels of miR-146b-5p in different gastric cancer cells were higher than that in GES-1 cells(t=26.21,13.08,11.09,11.09,6.41,P<0.05).The expression level of miR-146b-5p in MGC803 was significantly lower after transferring miR-146b-5p inhibitor(t=9.14,P<0.05).The proliferation of MGC803 cells with miR-146b-5p inhibitor was remarkably weakened,the apoptosis rate was increased(t=3.73,9.55,P<0.05).Moreover,miR-146b-5p mimics significantly reduced the luciferase activity of WT-NAIF1(t=8.35,P<0.05).The results suggested that miR-146b-5p mimics significantly down-regulated the expression of NAIF1 in MGC803 cells,while miR-146b-5p inhibitor up-regulated the expression of NAIF1(t=5.67,4.32,P<0.05).Transfection of miR-146b-5p on the basis of overexpression of NAIF1 significantly increased the proliferation and decreased the apoptosis rate(t=6.54,4.73,P<0.05).Conclusion MiR-146b-5p inhibit the cells apoptosis of gastric cancer through targeting NAIF1.