miR-876-5p通过负反馈调节上皮-间质样转化在胃癌进展中的作用及机制研究

The Role and Mechanism of miR-876-5p in the Progression of Gastric Cancer by Regulating Epithelial-Mesenchymal Transition through Negative Feedback

目的:探究miR-876-5p介导胃癌上皮-间质样转化(epithelial-mesenchymal transition,EMT)对胃癌进展的影响及机制。方法:通过实时荧光定量PCR法测定胃癌组织及癌旁正常组织内miR-876-5p表达差异;将胃癌细胞分为对照组、EMT组、miR-NC+EMT组、miR-876-5p+EMT组、miR-876-5p+EMT+异丙肾上腺素盐酸盐(isoproterenol hydrochloride,ISO)组,按照分组将miR-NC或miR-876-5p转染至胃癌细胞、5ng/mL转化生长因子-β(transforming growth factor-β1,TGF-β1)诱导24h以建立胃癌细胞EMT模型以及20μmoL/L细胞外信号调节激酶(extracellular signal-regulated protein kinase,ERK)信号通路激活剂ISO处理胃癌细胞24 h;处理结束后,免疫荧光染色检测各组细胞E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)的表达,蛋白质免疫印迹(Western blot)测定各组细胞内E-cadherin、Vimentin、N-cadherin、锌指蛋白转录因子(Snail)、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)及MMP-9蛋白表达,CCK-8法和EdU染色检测各组细胞增殖,Transwell小室法测定各组细胞迁移数目与侵袭数目,Western blot测定各组细胞内ERK信号通路相关蛋白表达。结果:胃癌组织中miR-876-5p相对表达量较癌旁正常组织下降(P<0.05);与EMT组比较,miR-876-5p+EMT组胃癌细胞E-cadherin荧光表达增强、Vimentin和N-cadherin荧光表达减弱,E-cadherin蛋白相对表达量上调而Vimentin、N-cadherin、Snail、MMP-2、MMP-9蛋白相对表达量均下调(P<0.05),细胞增殖活性和EdU阳性率降低(P<0.05),迁移数目与侵袭数目减少(P<0.05),p-ERK1/2蛋白相对表达量下调(P<0.05);而与miR-876-5p+EMT组比较,miR-876-5p+EMT+ISO组E-cadherin荧光表达减弱、Vimentin和N-cadherin荧光表达增强,E-cadherin蛋白相对表达量下调,Vimentin、N-cadherin、Snail、MMP-2、MMP-9蛋白相对表达量均上调(P<0.05),细胞增殖活性和EdU阳性率升高(P<0.05),迁移数目与侵袭数目增加(P<0.05),同时p-ERK1/2蛋白相对表达量上调(P<0.05)。结论:miR-876-5p通过调节胃癌细胞EMT进程进而影响肿瘤进展,该作用可能与抑制TGF-β1介导的ERK信号通路激活有关。

Objective:To explore the effect and mechanism of miR-876-5p regulating epithelial-mesenchymal transition(EMT)on the progression of gastric cancer.Methods:The differences in miR-876-5p expression in gastric cancer tissues and normal tissues adjacent to the cancer were determined by real-time fluorescence quantitative PCR;gastric cancer cells were divided into control group,EMT group,miR-NC+EMT group,miR-876-5p+EMT group,miR-876-5p+EMT+isoproterenol hydrochloride(ISO)group,miR-NC or miR-876-5p were transfected into gastric cancer cells according to the grouping,and 5 ng/mL transforming growth factor-β(TGF-β1)was induced for 24 h to establish an EMT model of gastric cancer cells and 20μmol/L extracellular.After treatment,the expression of E-cadherin,N-cadherin and vimentin was detected by immunofluorescence staining in each group of cells,and the expression of E-cadherin,Vimentin,N-cadherin,matrix metalloproteinase-2(MMP-2)and MMP-9 protein was determined by protein immunoblotting(Western blot)in each group of cells.The expression of E-cadherin,Vimentin,N-cadherin,Snail,matrix metalloproteinase-2(MMP-2)and MMP-9 in each group of cells was determined by western blot.Western blot was performed to determine the expression of ERK signaling pathway-related proteins in each group of cells.Results:The relative expression of miR-876-5p was decreased in gastric cancer tissues compared with normal tissues next to cancer(P<0.05);compared with the EMT group,the fluorescence expression of E-cadherin was enhanced and that of Vimentin and N-cadherin was decreased in the miR-876-5p+EMT group of gastric cancer cells,and the relative expression of E-cadherin protein was upregulated while Vimentin,N-cadherin,Snail,MMP-2,and MMP-9 protein relative expression were all downregulated(P<0.05),cell proliferation activity and EdU positivity were reduced(P<0.05),migration number and invasion number were decreased(P<0.05),and p-ERK1/2 protein relative expression was downregulated(P<0.05);while with miR-876-5p+EMT group,E-cadherin fluorescence expression was diminished,Vimentin and N-cadherin fluorescence expression was enhanced,E-cadherin protein relative expression was down-regulated,and Vimentin,N-cadherin,Snail,MMP-2,MMP-9 protein relative expression were upregulated(P<0.05),cell proliferation activity and EdU positivity were increased(P<0.05),migration number and invasion number were increased(P<0.05),while p-ERK1/2 protein relative expression was upregulated(P<0.05).Conclusion:miR-876-5p affects tumor progression by regulating the EMT process in gastric cancer cells,and this effect may be related to the inhibition of TGF-β1-mediated activation of the ERK signaling pathway.