DTX2通过Notch2/Akt轴促进结直肠癌细胞的迁移和侵袭

DTX2 overexpression promotes migration and invasion of colorectal cancer cells through the Notch2/Akt axis

目的探讨DTX2对结直肠癌(CRC)细胞迁移侵袭的影响及作用机制。方法利用基因工具干预CRC细胞,分为敲低组(DTX2-shRNA)、敲低空载组(neg-shRNA)、未转染组(con)、过表达空载组(pcDNA)及过表达组(pcDNA-DTX2),利用qRTPCR及Western blotting法检测转染效率。采用划痕和Transwell侵袭实验检测DTX2基因的表达改变对CRC细胞迁移侵袭能力的影响,并通过Western blotting检测各组中Notch2、NICD、Akt、p-AKT、MMP-2及MMP-9蛋白的表达水平。CRC细胞共转染pcDNA-DTX2和Notch2 siRNA,检测回复实验对CRC细胞迁移侵袭能力的影响。结果CRC细胞中DTX2 mRNA和蛋白表达量,敲低组中明显降低(P<0.01),过表达组中明显升高(P<0.01)。划痕和Transwell侵袭实验提示,CRC细胞迁移侵袭能力,DTX2 shRNA显著低于con和neg-shRNA(P<0.01);pcDNA-DTX2显著高于con和pcDNA(P<0.01)。Western blotting检测提示,Notch2、NICD、p-Akt及MMP-2蛋白平均表达水平,与con和neg-shRNA相比,DTX2 shRNA明显降低(P<0.05);与con和pcDNA相比,pcDNA-DTX2明显升高(P<0.05),而这些蛋白在con、neg-shRNA及pcDNA间的平均表达水平无明显差异(P>0.05)。回复实验提示,siRNA-Notch2可扭转pcDNA-DTX2增强的CRC细胞迁移侵袭的能力(P<0.01),相应蛋白水平的变化也具有统计学意义。结论DTX2通过Notch2/Akt轴促进CRC细胞迁移侵袭的能力,DTX2可能成为CRC的新型生物学指标。

Objective To investigate the effect of changes in DTX2 expression level on migration and invasion of colorectal cancer(CRC)cells and explore the mechanism.Methods Two CRC cell lines SW620 and LoVo were transfected with a specific shRNA targeting DTX2(DTX2-shRNA)or a DTX2-overexpressing plasmid(pcDNA-DTX2),and the transfection efficiency was evaluated with RT-qPCR and Western blotting.Scratch and Transwell assays were used to assess the changes in migration and invasion ability of the transfected cells,and the cellular expression levels of Notch2,NICD,AKT,p-Akt and MMP-2/9 proteins were detected with Western blotting.The CRC cells were co-transfected with pcDNA-DTX2 and Notch2 siRNA to assess the effect of Notch2 knockdown on DTX2 overexpression-induced enhancement of cell migration and invasion.Results The expression levels of DTX2 at both the mRNA and protein levels were significantly decreased in CRC cells transfected with DTX2-shRNA(P<0.01)and increased in cells transfected with pcDNA-DTX2(P<0.01).Scratch and Transwell assays showed that the migration and invasion abilities of CRC cells were significantly lowered following DTX2 knockdown(P<0.01)and were enhanced in cells with DTX2 overexpression(P<0.01).The expression levels of Notch2,NICD,p-Akt and MMP-2 proteins decreased significantly in CRC cells with DTX2 knockdown(P<0.05)and increased obviously in DTX2-overexpressing cells(P<0.05).In both of the two CRC cell lines,transfection with Notch2 siRNA obviously reversed the effect of DTX2 overexpression in promoting cell migration and invasion(P<0.01)and expressions of the related proteins.Conclusion DTX2 overexpression promotes migration and invasion of CRC cells through the Notch2/Akt axis,suggesting the potential of DTX2 as a new biological indicator of CRC.