长链非编码RNA LINC00886在胃贲门腺癌中的表达及其对胃癌细胞生物学行为的影响

Expression of long non-coding RNA LINC00886 in gastric cardia adenocarcinoma and its effects on biological characteristics of gastric cancer cells

目的:检测长链非编码RNA(long non-coding RNAs,LncRNAs)LINC00886在胃贲门腺癌(gastric cardia adenocarcinoma,GCA)组织及人胃癌细胞系中的表达及其对胃癌细胞体外增殖、迁移及侵袭等生物学行为的影响,并探讨其与上皮-间质转化(epithelial-mesenchymal transition,EMT)进程的相关性。方法:应用实时荧光定量PCR法检测LINC00886在GCA组织及其相应癌旁正常组织及人胃癌细胞系中的表达情况。采用脂质体法将携带有LINC00886的重组质粒pcDNA3.1-LINC00886转入胃癌SGC-7901和BGC-823细胞,用实时荧光定量PCR法检测转染效率。随后,采用MTS法和平板克隆形成实验、划痕愈合实验和Transwell小室迁移实验、Transwell小室侵袭实验分别检测LINC00886过表达对人胃癌细胞增殖、迁移及侵袭能力的影响。采用实时荧光定量PCR法和蛋白质印迹法检测pcDNA3.1-LINC00886转入SGC-7901和BGC-823细胞后EMT相关标志物E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)与波形蛋白(vimentin)mRNA和蛋白表达水平的变化。结果:在GCA组织中LINC00886的表达水平明显低于癌旁正常组织(P<0.01),且与肿瘤的TNM分期相关(P<0.05)。LINC00886在胃癌细胞中的表达均低于对照组(P值均<0.01)。与对照组相比,转染pcDNA3.1-LINC00886质粒的SGC-7901和BGC-823细胞中LINC00886的表达水平均明显上调(P<0.05或P<0.01);LINC00886过表达可抑制SGC-7901和BGC-823细胞的体外增殖能力(P值均<0.01);划痕愈合实验检测结果显示,LINC00886过表达后SGC-7901细胞的横向迁移能力受到抑制(P<0.05),但BGC-823细胞的横向迁移能力无变化(P>0.05);Transwell小室迁移实验检测结果显示,LINC00886过表达能抑制SGC-7901及BGC-823细胞的纵向迁移能力(P<0.05和P<0.01)。过表达LINC00886可上调SGC-7901和BGC-823细胞中E-cadherin mRNA(P<0.05和P<0.01)和蛋白的表达水平,降低N-cadherin和vimentin mRNA(P<0.05或P<0.01)和蛋白的表达水平。结论:LINC00886低表达可能与胃贲门腺癌的发生和发展相关。LINC00886过表达可以抑制胃癌细胞的体外增殖、迁移与侵袭能力,且可能参与EMT进程。

Objective:To study the expression status of long non-coding RNA(LncRNA)LINC00886 in human gastric cardia adenocarcinoma(GCA)tissues and gastric cancer cell lines,as well as its biological effects on the proliferation,migration and invasion of gastric cancer cells,and to explore its relationship with the epithelial-mesenchymal transition(EMT)process.Methods:Real-time fluorescent quantitative PCR was applied to detect the expression of LINC00886 in GCA tissues and the corresponding adjacent normal tissues as well as human gastric cancer cell lines.The liposome method was used to transfer the recombinant plasmid pcDNA3.1-LINC00886 carrying LINC 00886 gene into gastric cancer SGC-7901 and BGC-823 cells,and the transfection efficiency was detected by real-time fluorescent quantitative PCR.Then the effects of LINC00886 overexpression on the proliferation,migration and invasion abilities of SGC-7901 and BGC-823 cells were detected by MTS assay,clone formation assay,wound healing assay,Transwell migration and invasion assays,respectively.The mRNA and protein levels of EMT markers E-cadherin,N-cadherin and vimentin in SGC-7901 and BGC-823 cells transfected with pcDNA3.1-LINC00886 were detected by real-time fluorescent quantitative PCR and Western blotting,respectively.Results:The expression level of LINC00886 in GCA tissues was significantly lower than that in the adjacent normal tissues(P<0.01),and was associated with TNM stage of tumor(P<0.05).The expression level of LINC00886 in gastric cancer cell lines was lower than that in the control group(all P<0.01).The expression level of LINC00886 was upregulated in SGC-7901 and BGC-823 cells transfected with pcDNA3.1-LINC00886 plasmid as compared with the control group(P<0.05,P<0.01).Compared with the control group,the overexpression of LINC00886 inhibited the proliferation and invasion abilities of SGC-7901 and BGC-823 cells(all P<0.01),and the horizontal migration ability of SGC-7901 cells was decreased(P<0.05),but the horizontal migration rate of BGC-823 cells had no significant change(P>0.05).The vertica migration of SGC-7901 and BGC-823 cells was inhibited after LINC00886 overexpression(P<0.05,P<0.01).Overexpression of LINC00886 up-regulated the mRNA(P<0.05,P<0.01)and protein levels of E-cadherin in SGC-7901 and BGC-823 cells,and down-regulated the mRNA(P<0.05,P<0.01)and protein levels of N-cadherin and vimentin.Conclusion:The low expression of LINC00886 may be related to the occurrence and development of GCA.The overexpression of LINC00886 maybe inhibit the proliferation,migration and invasion abilities of gastric cancer cells in vitro,and may participate in the EMT process.