微小RNA-143-3p靶向调控Kirsten鼠肉瘤病毒癌基因介导丝裂原活化蛋白激酶/细胞外调节蛋白激酶信号通路对胃癌细胞增殖侵袭及上皮间质转化的作用机制

Effect of microRNA-143-3p regulating V-Ki-ras2 kirsten rat sarcoma viral oncogene homolog on gastric cancer progression through regulating mitogen-activated protein kinase/extracellular regulated protein kinase signaling pathway

目的探讨miR-143-3p靶向调控Kirsten鼠肉瘤病毒癌基因(KRAS)介导丝裂原活化蛋白激酶(MAPK)/细胞外调节蛋白激酶(ERK)信号通路对胃癌细胞增殖和侵袭的作用机制。方法双荧光素酶报告基因实验验证miR-143-3p与KRAS基因的靶向关系。将购自上海中国科学院的胃癌细胞株转染分组,将筛选人胃癌细胞株按照不同转染构建分为6组,miR-143-3p mimic阴性对照(negative control,NC)组、miR-143-3p Inhibitor NC组、miR-143-3p mimic组、miR-143-3p Inhibitor组、si-KRAS NC组、si-KRAS组、miR-143-3p Inhibitor+si-KRAS组。实时定量聚合酶链式反应(RT-qPCR)和蛋白质免疫印迹(Western blot)法检测转染后各组细胞相关因子的mRNA和蛋白的表达情况。细胞计数试剂盒-8(CCK-8)法及Transwell小室法检测胃癌细胞增殖及侵袭能力的变化。组间比较采用t检验。结果双荧光素酶报告基因实验证明KRAS是miR-143-3p靶基因。miR-143-3p mimic组的miR-143-3p表达量高于NC组(t=17.630,P<0.05),在miR-143-3p inhibitor组中表达量低于NC组(t=20.780,P<0.05),差异有统计学意义。在miR-143-3p mimic组(mRNA:KRAS为1.00±0.05比0.33±0.03,t=19.900;ERK为1.00±0.05比0.27±0.01,t=24.800;JNK为1.00±0.06比0.60±0.04,t=9.608;E-cadherin为1.00±0.04比1.69±0.06,t=16.570;N-cadherin为1.00±0.03比0.54±0.04,t=15.930;Snail为1.00±0.05比0.39±0.03,t=18.120;蛋白:KRAS为0.54±0.04比0.23±0.01,t=13.020;ERK为0.63±0.04比0.30±0.01,t=13.860;JNK为0.71±0.05比0.33±0.02,t=12.220;E-cadherin为0.85±0.05比1.20±0.06,t=7.762;N-cadherin为0.44±0.03比0.36±0.02,t=3.843;Snail为0.50±0.03比0.25±0.01,t=13.690)中KRAS、p-ERK/ERK、p-JNK/JNK、N-cadherin和Snail的mRNA和蛋白表达量低于NC组,而E-cadherin的mRNA和蛋白表达量高于NC组,细胞增殖(miR-143-3p mimic组:48 h为1.124±0.060比0.978±0.050,t=3.238;72 h为2.545±0.120比2.122±0.110,t=4.501;si-KRAS组:48 h为1.140±0.060比1.020±0.060,t=2.449;72 h为2.500±0.120比2.070±0.100,t=4.768)及侵袭能力(miR-143-3p mimic组:210.00±10.00比110.00±7.00,t=14.190;si-KRAS组:215.00±12.00比100.00±6.00,t=14.850)低于NC组,差异均有统计学意义(P值均<0.05);而miR-143-3p Inhibitor组前述趋势逆转(P值均<0.05),差异有统计学意义。结论miR-143-3p抑制靶向KRAS基因表达,阻断MAPK/ERK信号通路的激活,从而抑制人胃癌增殖侵袭,并调控上皮间质转化进程。

Objective To investigate the effect of miR-143-3p on the proliferation and invasion of gastric cancer cells by targeting kirsten rat sarcoma(KRAS)gene and mediating mitogen-activated protein kinase(MAPK)/extracellular regulated protein kinase(ERK)signaling pathway.Methods Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-143-3p and KRAS gene.Gastric cancer cells purchased from Chinese Academy of Sciences in Shanghai were transfected into different groups.Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)and Western blotting were used to detect the mRNA and protein expressions of related indexes.Cell counting kit-8(CCK-8)and Transwell assays were used to examine the proliferation and invasion of gastric cancer cells.Results Dual luciferase reporter gene assay verified that KRAS was the target gene of miR-143-3p.After cell grouping and transfection,as compared with corresponding negative control(NC)group,miR-143-3pmimic group showed increased miR-143-3p expression(t=17.630),which was decreased in miR-143-3p inhibitor group(t=20.780;all P<0.05).In miR-143-3p mimic group and si-KRAS group,there were increased mRNA for miR-143-3p mimic group,KRAS:1.00±0.05 vs.0.33±0.03,t=19.900;ERK:1.00±0.05 vs.0.27±0.01,t=24.800;JNK:1.00±0.06 vs.0.60±0.04,t=9.608;E-cadherin:1.00±0.04 vs.1.69±0.06,t=16.570;N-cadherin:1.00±0.03 vs.0.54±0.04,t=15.930;Snail:1.00±0.05 vs.0.39±0.03,t=18.120.Expression levels of KRAS,p-ERK/ERK,p-JNK/JNK,N-cadherin and Snail,while decreased E-cadherin mRNA and protein expression levels,decreased cell proliferation(formiR-143-3p mimic group at 48 h:1.124±0.060 vs.0.978±0.050,t=3.238;at 72 h:2.545±0.120 vs.2.122±0.110,t=4.501.for si-KRAS group at 48 h:1.140±0.060 vs.1.020±0.060,t=2.449;at 72 h:2.500±0.120 vs.2.070±0.100,t=4.768)and invasion(for miR-143-3p mimic group:210.00±10.00 vs.110.00±7.00,t=14.190;for si-KRAS group:215.00±12.00 vs.100.00±6.00,t=14.850),and the differences were statistically significant(all P<0.05).While miR-143-3p inhibitor group had the opposite trends mentioned above(all P<0.05).Conclusion MiR-143-3p inhibits the expression of targeted KRAS gene and blocks the activation of MAPK/ERK signaling pathway,thus inhibiting the proliferation and invasion of human gastric cancer cells and regulating epithelial mesenchymal transition process.