LINC00680靶向miR-431-5p调控胃癌细胞增殖、上皮间充质转化、迁移和侵袭

LINC00680 regulates proliferation, epithelial-mesenchymal transition,migration and invasion of gastric cancer cells by targeting miR-431-5p

目的:研究LINC00680对胃癌细胞增殖、上皮间充质转化(EMT)、迁移和侵袭的影响,并探讨其可能机制。方法:实时荧光定量PCR(RT-qPCR)检测胃癌细胞系(SNU-1、AGS、HS-746T)和人胃黏膜上皮永生细胞系GES1中LINC00680和miR-431-5p表达水平。双荧光素酶报告实验和RT-qPCR确定LINC00680对miR-431-5p的调控作用。采用脂质体转染法将siNC、si-LINC00680、miR-NC、miR-431-5p mimics、si-LINC00680+anti-miR-NC、si-LINC00680+anti-miR-431-5p分别转染AGS细胞。细胞计数试剂盒(CCK-8)检测细胞活力;Transwell实验检测细胞迁移和侵袭;Western blot检测细胞周期素D1(CyclinD1)、基质金属蛋白酶(MMP2)、MMP9、上皮细胞钙黏蛋白(E-Cadherin)、神经钙黏素(N-Cadherin)和波形蛋白(Vimentin)蛋白表达。结果:与GES1比较,胃癌细胞系中LINC00680表达水平显著升高,miR-431-5p表达显著降低(P<0.05)。LINC00680靶向负调控miR-431-5p表达。抑制LINC00680或过表达miR-431-5p均可显著降低AGS细胞的增殖、迁移和侵袭能力,降低CyclinD1、MMP2和MMP9蛋白的表达水平,且抑制miR-431-5p表达可逆转si-LINC00680对AGS增殖、迁移和侵袭的抑制作用(P<0.05)。抑制LINC00680可显著降低AGS细胞N-Cadherin和Vimentin蛋白表达水平,增加E-Cadherin蛋白表达水平,且抑制miR-431-5p表达可逆转si-LINC00680对AGS细胞E-Cadherin、N-Cadherin、Vimentin表达的影响(P<0.05)。结论:抑制LINC00680表达可降低胃癌细胞的增殖、迁移和侵袭能力,抑制EMT进程,其机制与靶向调控miR-431-5p表达有关。

Objective:To study the effect of LINC00680 on the proliferation,epithelial mesenchymal transition(EMT),mi-gration and invasion of gastric cancer cells,and further to explore its possible mechanism.Methods:The expression levels of LINC00680 and miR-431-5p in gastric cancer cell lines(SNU-1,AGS,HS-746T)and human gastric mucosal epithelial immortal cell line GES1 were detected by Real-time quantitative PCR(RT-qPCR).Dual luciferase report experiment and RT-qPCR were performed to confirm the relationship between LINC00680 and miR-431-5p.si-NC,si-LINC00680,miR-NC,miR-431-5p mimics,siLINC00680+anti-miR-NC,si-LINC00680+anti-miR-431-5p were transfected into AGS cells by lipofection.Cell counting kit(CCK-8)was used to test cell viability;Transwell test was applied to detect cell migration and invasion;the expression of CyclinD1,matrix me-talloproteinase(MMP2),MMP9,epithelial cadherin(E-Cadherin),N-Cadherin and Vimentin proteins was measured by Western blot.Results:Compared with GES1,the expression of LINC00680 was significantly increased,the expression of miR-431-5p was sig-nificantly reduced in gastric cancer cell lines(P<0.05).LINC00680 negatively regulates the expression of miR-431-5p.LINC00680 inhibition or miR-431-5p overexpression can significantly reduce the proliferation,migration and invasion ability of AGS cells,as well as reduce the expression level of CyclinD1,MMP2 and MMP9 proteins,and miR-431-5p inhibition can reverse the inhibitory effect of si-LINC00680 on proliferation,migration and invasion of AGS cells(P<0.05).Inhibition of LINC00680 can significantly reduce the expression levels of N-Cadherin and Vimentin proteins in AGS cells,as well as increase the expression level of E-Cadherin protein,and miR-431-5p inhibition can reverse the effect of si-LINC00680 on the expression of E-Cadherin,N-Cadherin and Vimentin in AGS cells(P<0.05).Conclusion:Inhibiting LINC00680 can reduce the proliferation,migration and invasion ability of gastric cancer cells and inhibit the EMT process,and its mechanism is correlated to the targeted regulation of miR-431-5p expression.