目的 探讨转录延伸样因子7(TCEAL7)对低分化人胃癌细胞株BGC823侵袭、迁移的影响及其机制.方法 应用实时定量聚合酶链反应(Real-time PCR)和Western blot法检测TCELA7 mRNA和蛋白在BGC823和正常胃黏膜细胞株GES-1中的表达;构建TCEAL7真核表达载体GFP-TCEAL7并转染BGC823细胞48 h,检测转染效果;噻唑蓝(MTT)法检测GFP-TCEAL7转染对BGC823细胞活性的影响;划痕实验、Transwell小室实验检测细胞侵袭、迁移;应用Real-time PCR和Western blot法检测转染GFP-TCEAL7转染对BGC823细胞侵袭、迁移基因表达水平的影响.结果 Real-time PCR和Western blot法显示,BGC823的TCEAL7表达(mRNA:0.280±0.048,蛋白:0.181±0.038)明显低于GES-1(mRNA:0.474±0.062,蛋白:0.346±0.057;t=-6.061,P=0.000;t=-5.900,P=0.000).GFP-TCEAL7转染后胃癌BGC823细胞TCEAL7的mRNA及蛋白表达(1.886±0.326、1.007±0.262)比对照组(0.302±0.053、0.172±0.030)显著上调(t=11.748,P =0.000:t=7.756,P=0.000).MTT结果显示,转染了GFP-TCEAL7的BGC823细胞活性(0.672±0.083)比对照组(1.242±0.215)显著减弱(t=-6.058,P =0.000).划痕实验、Transwell小室实验显示转染了GFP-TCEAL7的BGC823细胞迁移能力[转染组(53.945 ±9.952)%,对照组(81.017±7.208)%]和侵袭能力(转染组76.333±12.420,对照组121.167±11.771)均明显降低(t=-5.396,P =0.000;t=-6.418,P=0.000).Real-time PCR和Western blot法显示转染GFP-TCEAL7的BGC823细胞基质金属蛋白酶(MMP)-7 、MMP-9的mRNA和蛋白表达水平下降,组织基质金属蛋白酶抑制剂-1 (TIMP1)和E-钙黏蛋白(E-cadherin)表达明显增高.结论 过表达TCEAL7可抑制BGC823细胞的侵袭、迁移.
Objective To explore the effects and mechanism of transcription elogation factor A (S Ⅱ) like 7 (TCEAL7) on invasion and migration of poorly differentiated human gastric cancer cell line BGC823 in vitro.Methods Real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting were utilized to detect mRNA and protein expression of TCEAL7 in BGC823 cells and normal gastric mucosal cell line GES-1.TCEAL7 eukaryotic expression vector was designed and transfected into BGC823 cells for 48 h.The methyl thiazol tetrazolium (MTT) assay was applied to test the cell activity affected by GFP-TCEAL7 transfection.The wound assay and Transwell chamber assay were employed to detect the invasion and migration of cells.Real-time PCR and Western blotting were used to detect the mRNA and protein expression of invasion-and migration-related genes.Results Real-time PCR and Western blotting demonstrated that the expression of TCEAL7 was significantly wearker in BGC823 cells (mRNA:0.280 ± 0.048;protein:0.181 ± 0.038) than GES-1 cells (mRNA:0.474 ± 0.062;protein:0.346 ± 0.057;t =-6.061,P =0.000;t =-5.900,P =0.000).The expression of TCEAL7 was up-regulated significantly in BGC823 cells after GFP-TCEAL7 transfection (1.886 ± 0.326,1.007 ± 0.262) as compared with control group (0.302 ± 0.053,0.172 ± 0.030;t =11.748,P =0.000;t =7.756,P =0.000).Result of MTT exhibited that the proliferation inhibition rate increased significantly in BGC823 cells after GFP-TCEAL7 transfection (0.672 ±0.083) as compared with control group (1.242 ±0.215) (t =-6.058,P =0.000).Wound assay and Transwell chamber assay revealed that GFP-TCEAL7 transfection induced decreased migration [for transfection group,(53.945 ± 9.952) %;for control group,(81.017 ± 7.208)%] and invasion [for transfection group,(76.333 ± 12.420);for control group,(121.167 ± 11.771)] of cells (t=-5.396,P=0.000;t=-6.418,P=0.000).The results of Real-time PCR and Western blotting showed that the mRNA and protein levels of matrix metalloproteinase (MMP)-7 and MMP-9 were significantly down-regulated by TCEAL7 overexpression,while the mRNA and protein expression levels of TIMP1 and E-cadherin up-regulated significantly.Conclusion Overexpression of TCEAL7 could inhibit invasion and migration of BGC823 cells.