异位表达LncRNA PVT1/EIF4A1促进胃癌细胞增殖迁移的作用研究

Study on the effect of ectopic expression of LncRNA PVT1/EIF4A1 on the proliferation and migration of gastric cancer cells

目的探索长链非编码RNA人浆细胞瘤转化迁移基因1(plasmacytoma variant translocation 1,PVT1)协同真核翻译起始因子4A1(human eukaryotic translation initiation factor4A1,EIF4A1)促进人胃癌细胞SGC-7901增殖与迁移能力的作用。方法使用Western Blot检测EIF4A1、real time-PCR检测PVT1在胃癌细胞(SGC-7901、NCI-N87、MGC-803、BGC-823)和正常胃黏膜细胞(GES-1)中的表达量。单独或共同过表达胃癌细胞SGC-7901中的PVT1和EIF4A1,EIF4A1的蛋白表达量使用免疫印迹法检测,PVT1的表达水平使用real time-PCR检测,采用划痕实验和Transwell实验检验细胞的迁移能力,采用MTT实验和平板克隆形成法检验细胞的增殖情况,采用免疫印迹法检测E-cadherin,N-cadherin,smad3和TGF-β蛋白表达量。结果以正常胃黏膜细胞作为对照,筛选有统计学差异的PVT1和EIF4A1的本底表达量低的胃癌细胞(P<0.05)。单独或共同过表达胃癌细胞中PVT1和EIF4A1后,共同过表达组平板克隆形成数为492±8.72,单独过表达PVT1组平板克隆形成数为251±1.53,单独过表达EIF4A1组平板克隆形成数为228±1.52,对照组平板克隆形成数为205±2.08。在MTT实验中,共同过表达组OD值高于单独过表达组和对照组。Transwell结果显示共同过表达组迁移细胞数为308±29.10,单独过表达PVT1组迁移细胞数为222±14.42,单独过表达EIF4A1组迁移细胞数为206±10.58,对照组迁移细胞数为169±18.04。划痕实验结果提示共同过表达组伤痕愈合速率高于单独过表达组和对照组。免疫印迹结果说明共同过表达组E-cadherin低于单独过表达组和对照组,共同过表达组N-cadherin、smad3和TGF-β高于单独过表达组和对照组。以上结果均具有统计学差异(P<0.05)。结论过表达PVT1和EIF4A1可以协同促进SGC-7901细胞的增殖能力,并通过改变EMT相关蛋白的表达促进胃癌细胞SGC-7901的迁移能力。

Objective Explore the effect of long non-coding RNA human plasmacytoma variant translocation 1(PVT1)and human eukaryotic translation initiation factor4A1(EIF4A1)on the proliferation and migration of gastric cancer(GC)cell SGC-7901.Methods Used Western Blot to detect EIF4A1 and real time-PCR to detect PVT1 in GC cells(SGC-7901,NCI-N87,MGC-803,BGC-823)and normal gastric mucosal cells(GES-1).Ectopic expressed PVT1 and EIF4A1 in GC cell SGC-7901 individually or together.Used Western Blot to detect the protein expression of EIF4A1.And used real time-PCR to detect the expression level of PVT1.Detected the migration ability of cells by the scratch test and the transwell assay.The proliferative ability of the cells was detected by the MTT and the clone formation assay.And the protein level of E-cadherin,N-cadherin,smad3 and TGF-βwas detected by Western Blot.Results GC cells with low background expression of PVT1 and EIF4A1 were screened,and there was a statistical difference(P<0.05).After ectopic expression of PVT1 and EIF4A1 individually or jointly,the number of plate clone formation in the co-ectopic expression group was 492±8.72.The number of plate colonies formed in the PVT1 ectopic expression group alone was 251±1.53.And that in the EIF4A1 ectopic expression group and control group was 228±1.52 and 205±2.08 respectively.In MTT assay,the OD value of co-ectopic expression group was higher than that of individual ectopic expression group and control group.In the transwell assay,the number of migrated cells of the co-ectopic expression group was 308±29.10,and in the PVT1 ectopic expression group,EIF4A1 ectopic expression group and control group was 222±14.42,206±10.58,and169±18.04 respectively.Cell scratch tests showed the wound healing rate in the co-ectopic expression group was higher than that in the individual ectopic expression group and the control group.The results of Western Blot showed that E-cadherin in the ectopic expression group was lower than that in the ectopic expression group and control group.N-cadherin,smad3 and TGF-βin the ectopic expression group were higher than those in the individual ectopic expression group and control group.All of these results were statistically significant(P<0.05).Conclusion Ectopic expression of PVT1 and EIF4A1 can promote the proliferation ability and migration ability of SGC-7901 cells by altering the expression of EMT related proteins.