DAB2IP稳定细胞系的构建及其对结直肠癌HT29细胞侵袭迁移能力的影响

Construction of DAB2IP stable cell line and its effect on invasion and migration of HT29 cells

目的构建DAB2IP高表达的结直肠癌HT29稳定细胞系,检测其对HT29细胞侵袭及迁移能力的影响。方法将携带DAB2IP基因序列的pcDNA3.1质粒转染HT29细胞,24h后加入适量浓度的G418筛选(10μg/mL),两周后,通过实时定量PCR(qPCR)和Westernblot方法检测DAB2IPmRNA水平和蛋白水平,验证DAB2IP的高表达;进一步,通过Transwell及划痕实验检测DAB2IP对HT29细胞侵袭及迁移能力的影响;最后,通过Westernblot方法检测其对E-cadherin和vimentin蛋白的表达,初步探讨DAB2IP影响HT29细胞侵袭迁移能力的机制。结果经G418筛选后,DAB2IP的mRNA和蛋白水平均较对照组明显升高;高表达DAB2IP能够明显抑制HT29细胞的侵袭及迁移能力,差异具有统计学意义(P<0.001);高表达DAB2IP能够促进HT29细胞E-cadherin蛋白的表达,抑制vimentin蛋白的表达。结论成功筛选出DAB2IP高表达的HT29稳定细胞系,高表达DAB2IP能够通过抑制细胞上皮间质转化过程从而抑制HT29细胞侵袭及迁移能力。

Objective The aim of this study was to construct the stable cell line of colorectal cancer HT29 cells with high expression of DAB2IP and its effect on invasion and migration of HT29 cells.Methods The pcDNA3.1 plasmid carrying the DAB2IP gene sequences was transfected into HT29 cells.After 24 hours,10 μg/mL of G418 was added the cells to screen the cells with high expression of DAB2IP.Two weeks later,DAB2IP at levels of mRNA and protein in screened cells was detected to verify high expression of DAB2IP by real-time quantitative PCR(RT-qPCR)and Western blot.Further,the effects of DAB2IP on the invasion and migration of HT29 cells were detected by Trans well and scratch assays.Finally,the expression of E-cadherin and vimentin proteins was detected to explore the mechanism of DAB2IP affecting the invasion and migration ability of HT29 cells by Western blot.Results After screening with G418,the mRNA and protein levels of DAB2IP were significantly higher than those of the control cells.Over-expression of DAB2IP could significantly inhibit the invasion and migration of HT29 cells;the difference was statistically significant( P <0.001).Over-expression of DAB2IP could promote the expression of E-cadherin protein and inhibit the expression of vimentin protein in TH29 cells.Conclusion The HT29 stable cell line with high expression of DAB2IP was successfully screened.Over-expression of DAB2IP can inhibit the invasion and migration in HT29 cells by inhibiting the process of cell epithelial-mesenchymal transition(EMT).