miR-122-5p靶向ADAM10调控Notch信号通路对人卵巢癌细胞SKOV-3的上皮-间质转化的抑制作用  

Inhibition effect of miR-122-5p targeted ADAM10 regulates Notch signaling pathway on epithelial-mesenchymal transformation in human ovarian cancer SKOV-3 cells

作　　者：李根林 刘杰[1] 谢晶[1] 余芙蓉[1] 胡意 Li Genlin;Liu Jie;Xie Jing(Dept of Obstetrics and Gynecology,The First Affiliated Hospital of University of South China,Hengyang 421000)

机构地区：[1]南华大学附属第一医院妇科,衡阳421000

出　　处：《安徽医科大学学报》2021年第1期54-59,共6页Acta Universitatis Medicinalis Anhui

基　　金：湖南省教育厅科学研究项目(编号:17C1386)。

摘　　要：目的研究miR-122-5p对人卵巢癌细胞SKOV-3上皮-间质转化(EMT)的影响,阐明其可能机制。方法常规体外培养人卵巢癌细胞SKOV-3,将miR-122-5p模拟物(miR-122-5p mimic)及其阴性对照(mimic NC)转染至SKOV-3细胞,分为空白对照组(Blank组)、mimic NC组和miR-122-5p mimic组,采用实时荧光定量PCR(qRT-PCR)检测各组细胞miR-122-5p和ADAM10 mRNA的表达水平;Western blot检测各组细胞ADAM10蛋白的表达水平;生物信息学预测miR-122-5p与ADAM10靶向匹配,采用双荧光素酶报告系统验证两者在SKOV-3细胞中的靶向关系。再将miR-122-5p mimic和ADAM10过表达质粒(pcDNA-ADAM10)分别或同时转染至SKOV-3细胞中,分为空白对照组(Blank组)、miR-122-5p mimic组、ADAM10过表达组(pc-ADAM10组)和miR-122-5p mimic+pc-ADAM10组,采用Transwell检测SKOV-3细胞的迁移和侵袭能力;Western blot检测细胞Notch信号通路相关蛋白Notch1、HES1和HEY1,EMT相关蛋白Snail、E-cadherin和N-cadherin的表达水平。结果 miR-122-5p mimic组SKOV-3细胞中miR-122-5p的表达水平较mimic NC组的升高(P<0.01),表明miR-122-5p mimic稳定转染至SKOV-3细胞。miR-122-5p mimic组SKOV-3细胞中ADAM10 mRNA和蛋白的表达水平较mimic NC组的降低(P<0.01),双荧光素酶报告系统证实ADAM10是miR-122-5p靶基因。与Blank组比较,miR-122-5p mimic组SKOV-3细胞发生迁移和侵袭的数量降低(t=6.004,P<0.05;t=7.289,P<0.01),而pc-ADAM10组的升高(t=13.181,P<0.001;t=11.504,P<0.01);miR-122-5p mimic组细胞的Notch、HES1、HEY1、Snail和N-cadherin蛋白表达水平下降(P<0.05,P<0.01),而pc-ADAM10组的上升(P<0.01);miR-122-5p mimic组细胞的E-cadheirn蛋白表达水平上升(P<0.01),而pc-ADAM10组的下降(P<0.01)。与pc-ADAM10组比较,miR-122-5p mimic+pc-ADAM10能减弱pc-ADAM10的促EMT作用,且下调Notch信号通路活性。结论 miR-122-5p通过阻断Notch信号通路抑制人卵巢癌细胞SKOV-3的EMT,其机制可能与miR-122-5p靶向抑制ADAM10的表达有关。Objective To investigate the effects of miR-122-5 p on epithelial-mesenchymal transformation(EMT) in human ovarian cancer SKOV-3 cells, and to elucidate its possible mechanism.Methods Human ovarian cancer SKOV-3 cells were cultured in vitro and transfected with miR-122-5 p mimic and negative control(mimic NC). Then the cells were divided into blank control group(Blank), mimic NC group, and miR-122-5 p mimic group. The expression of miR-122-5 p and ADAM10 mRNA was detected by real-time fluorescence quantitative PCR(qRT-PCR). Western blot was used to detect the expression of ADAM10 protein. Bioinformatics predicted the target matching of mir-122-5 p and ADAM10, and dual luciferase reporter system was used to verify the target relationship. In addition, miR-122-5 p mimic and ADAM10-overexpressed plasmid(pcDNA-ADAM10) were respectively or simultaneously transfected into SKOV-3 cells, then the cells were divided into blank control group(Blank), miR-122-5 p mimic group, ADAM10 overexpression group(pc-ADAM10) and miR-122-5 p mimic+pc-ADAM10 group. Transwell was used to detect the migration and invasion ability of SKOV-3 cells. Western blot was used to detect the expression of Notch signaling pathway related proteins Notch1, HES1 and HEY1, EMT related proteins Snail, E-cadherin and N-cadherin.Results Compared with mimic NC group, the expression of miR-122-5 p of SKOV-3 cells in miR-122-5 p mimic group increased(P<0.01), indicating that miR-122-5 p mimic was stably transfected into SKOV-3 cells. Compared with mimic NC group, the expression of ADAM10 mRNA and protein of SKOV-3 cells in miR-122-5 p mimic group was lower(P<0.01). ADAM10 was confirmed as a target gene of miR-122-5 p by the dual luciferase reporter system. Compared with Blank group, the migration and invasion of SKOV-3 cells in miR-122-5 p mimic group decreased(t=6.004, P<0.05;t=7.289, P<0.01), while that of pc-ADAM10 group increased(t=13.181, P<0.001;t=11.504, P<0.01);the expression of Notch, HES1, HEY1, Snail and N-cadherin proteins were suppressed(P<0.05;P<

关 键 词：人卵巢癌细胞SKOV-3 上皮-间质转化 NOTCH信号通路 去整合素金属蛋白酶10 

分 类 号：R737.31[医药卫生—肿瘤]