Dach1表达下调促进结肠癌细胞SW480侵袭和迁移的机制研究

Down-regulation of Dach1 Enhances Ability of Invasion and Migration of Colon Cancer Cell Line SW480

目的研究Dach1表达下调对结肠癌细胞SW480侵袭迁移能力的影响及其可能机制。方法用脂质体将si-Dach1转染进SW480细胞株;Western blot检测细胞Dach1及EMT信号通路蛋白E-cadherin、β-catenin、Zeb1的表达水平,Real-time PCR检测Zeb1的mRNA表达水平,Transwell侵袭迁移实验以及划痕实验检测对照组SW480con及SW480si-Dach1组细胞侵袭数目和迁移距离。miR-200c mimics转染SW480si-Dach1细胞重复上述实验。结果 SW480si-Dach1组的miR-200c表达量为SW480con组的0.531倍,SW480si-Dach1组Zeb1的转录水平表达高于SW480con组[(2.21±0.35)vs.(1.01±0.02),P<0.01],SW480si-Dach1组Transwell小室实验48 h迁移的细胞数多于SW480con组[(102.33±1.53)vs.(44.67±2.52),P<0.01],SW480si-Dach1组穿透基质胶的细胞数多于SW480con组[(45.33±2.52)vs.(17.33±0.58),P<0.01],SW480si-Dach1组划痕实验24 h的平均距离大于SW480con组[(111.67±5.86)μm vs.(44.21±3.11)μm,P<0.01],上述实验在SW480con组与SW480si-Dach1+miR-200c组之间差异没有统计学意义。Western blot实验中SW480si-Dach1组较SW480con组Zeb1及β-catenin表达升高,E-cadherin表达下降,而这些蛋白表达变化可以被高miR-200c逆转。结论低表达Dach1能够通过抑制miR-200c从而上调Zeb1表达,进而激活EMT通路,最终正性调控SW480细胞的转移和迁移能力。

Objective To investigate the relationship between down-regulation of Dach1 and the ability of invasion and migration of colon cancer cell SW480,to discuss the mechanism of invasion and migration influenced by the expression of Dach1.Methods si-Dach1 were transfected into SW480 cell strain by lipidosome.Western blotting was used to detect cell Dach1,protein E-cadherin,β-catenin and Zeb1 in EMT signaling pathway.Real-time PCR was used to detect the mRNA expression of Zeb1.Transwell and wound healing assay were used to detect the migration and invasion of SW480 cells.Results The expression of miR-200 c in si-Dach1 group decreased to 0.531 time that in control group.The mRNA expression of Zeb1 was higher in si-Dach1 group than in control group[(2.21±0.35) vs.(1.01±0.02),P<0.01].The penetrated cells were more in the si-Dach1 group than in the control group in transwell assay[(102.33±1.53) vs.(44.67±2.52),P<0.01].And the same result was found in invasion assay[(45.33±2.52) μm vs.(17.33±0.58) μm,P<0.01].The average migrated distance was significantly longer in the si-Dach1 group than in the control group in wound healing assay[(111.67±5.86) μm vs.(44.21±3.11) μm,P<0.01].Over-expression of miR-200 c reversed the enhanced ability of invasion and migration caused by down-regulation of Dach1 in SW480.After down-regulation of Dach1,Western blotting showed that the protein level of Zeb1 and β-catenin was increased,while the protein level of E-cadherin was decreased,all these change reversed after over-expression of miR-200 c.Conclusion Down-regulation of Dach1 enhances the migration and invasion of SW480 cells by affecting the epithelial mesenchymal transition through inhibiting miR-200 c.