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

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ADAM9 mRNA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。
ABSTRACT: 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