miR-134-3p通过靶向调节ADAM9/EGFR/AKT抑制结肠癌细胞的活性和迁移  

miR-134-3p inhibits the activity and migration of colon cancer cells through targeting ADAM9/EGFR/AKT pathway

作　　者：付立芳 孙杰 FU Li-fang;SUN Jie(Chinese Medicine Hospital of Linyi City,Linyi 276000;Department of Traditional Chinese Medicine,Shandong Medical College,Linyi 276000,China)

机构地区：[1]临沂市中医医院,山东临沂276000 [2]山东医学高等专科学校中医学教研室,山东临沂276000

出　　处：《西安交通大学学报（医学版）》2018年第5期653-659,共7页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：山东省教育厅课题资助项目(No.J13LK56);山东省临沂市科技发展计划项目(No.201515053)~~

摘　　要：目的探究miR-134-3p是否通过靶向调控ADAM9/EGFR/AKT通路影响结肠癌细胞的存活和迁移。方法实时定量PCR检测正常结肠上皮细胞NCM460和结肠癌细胞SW480,HCT116和RKO中miR-134-3p的表达,Western免疫印迹检测上述细胞中ADAM9、EGFR和AKT的蛋白表达;miR-134-3p mimic/miR-134-3p inhibitor转染SW480细胞后,CCK-8试剂盒、流式细胞术和Transwell迁移分析法分别用来检测各组细胞的增殖、凋亡、迁移能力;Western免疫印迹检测各组细胞ADAM9/EGFR/AKT通路蛋白表达和其磷酸化水平;荧光素酶报告分析实验用于验证miR-134-3p和ADAM9的靶向关系。结果与NCM460细胞系相比,miR-134-3p在3种人结肠癌细胞系中低表达,差异具有统计学意义(P<0.05),而ADAM9、EGFR和AKT在结肠癌细胞系中表达升高2~3倍(P<0.05)。miR-134-3p mimic转染后SW480细胞的增殖能力降低,迁移细胞数目减少。miR-134-3p mimic转染后细胞凋亡率升高至(15.0±1.1)%,与对照组(9.0±1.7)%相比差异有统计学意义(P<0.05)。与对照组相比,miR-134-3p mimic转染后ADAM9、p-EGFR/EGFR和p-AKT/AKT的水平降低,差异均具有统计学意义(P<0.05)。荧光素酶报告实验证实miR-134-3p可靶向结合ADAM9mRNA而抑制其表达;而SW480细胞转染miR-134-3p inhibitor则与miR-134-3p mimic作用相反。结论 miR-134-3p通过靶向调控ADAM9的表达而抑制EGFR/AKT通路,从而抑制结肠癌细胞的存活和迁移。Objective To explore whether miR-134-3p regulates the survival and migration of colon cancer cells through targeting ADAM9/EGFR/AKT pathway.Methods Real-time qPCR was used for detecting the miR-134-3p expression in the normal colonic epithelia(NCM460)and colon cancer cells(SW480,HCT116 and RKO).Meanwhile,Western blot was used for measuring the protein expressions of ADAM9,EGFR and AKT in these cells.Subsequently,cells were transfected with miR-134-3p mimic/miR-134-3p inhibitor,cell proliferation,apoptosis and migration were examined by CCK-8 kits,flow cytometry and Transwell migration assays,respectively.Next,the protein expressions and phosphorylation of ADAM9,EGFR and AKT were measured to evaluate the activation of ADAM9/EGFR/AKT pathway.Finally,the relationship between miR-134-3p and ADAM9 was verified with luciferase assay.Results MiR-134-3p was significantly down-regulated in the three colon cancer cells compared to the normal colonic epithelia NCM460 cells(P<0.05).In contrast,the expressions of ADAM9,EGFR and AKT increased by 2-3 folds in cancer cells compared with NCM460 cells(P<0.05).The proliferation capability of SW480 cells decreased and the count of migrated cells reduced after miR-134-3p mimic transfection.Cell apoptosis percentage elevated to(15.0±1.1)%,which was significantly higher than that of the control(9.0±1.7)%(P<0.05).In addition,there was a significant decrease in ADAM9,p-EGFR/EGFR and p-AKT/AKT compared with the control(P<0.05).Luciferase assay confirmed that miR-134-3p could bind to ADAM9 mRNA to suppress its expression.However,miR-134-3p inhibitor showed the opposite effect against miR-134-3p mimic transfection in SW480 cells.Conclusion miR-134-3p inhibited survival and migration of colon cancer cells through targeting ADAM9 and the subsequent EGFR/AKT signaling.

关 键 词：结肠癌 miR-134-3p 金属蛋白酶解离素9 细胞增殖 凋亡 迁移 

分 类 号：Q291[生物学—细胞生物学] R34[医药卫生—基础医学]