重组腺病毒载体介导人LASS2基因在肝癌细胞转移中的抑制作用

Construction of LASS2 gene recombinant adenovirus and its effect on the inhibition of metastasis of hepatocellular carcinoma cell line HCCLM3

目的:探讨人源性长寿保障基因(homo-sapiens longevity assurance homologue 2 of yeast LAG1,LASS2)对人肝癌HCCLM3细胞体外转移的影响及可能的作用机制。方法:构建含人LASS2基因的重组腺病毒并转染入HCCLM3细胞;采用蛋白质印迹法检测HCCLM3细胞中LASS2蛋白的表达水平;划痕实验及体外侵袭实验检测LASS2基因过表达对HCCLM3细胞在迁移和侵袭等转移能力上的改变;免疫共沉淀法验证LASS2蛋白与V-ATPase(vacuolarH+-ATPase)质子泵中的c亚基(ATP6L)在细胞内相互结合的情况;通过对细胞内外H+浓度的测定,探讨LASS2过表达对V-ATPase功能抑制的情况。结果:成功构建了含人LASS2基因的重组腺病毒,LASS2基因在HCCLM3细胞中能有效表达;LASS2过表达可使HCCLM3细胞的侵袭和迁移能力均明显下降(P<0.01),其中侵袭能力下降达(48.3±7.6)%,穿过划痕的细胞个数从对照组的(59.00±6.87)个下降至(10.83±3.75)个;LASS2蛋白与ATP6L蛋白在HCCLM3细胞内特异性结合;LASS2过表达后,细胞内外H+浓度发生明显变化,表明V-ATPase质子泵功能受到抑制。结论:LASS2基因在HCCLM3细胞中过表达可抑制肝癌细胞的迁移和侵袭能力,其机制可能与LASS2蛋白结合ATP6L,抑制其分泌H+功能相关。

Objective： To investigate the effect of human homo-sapiens longevity assurance homologue 2 of yeast LAG1 （LASS2） gene on the metastasis of hepatocellular carcinoma cell HCCLM3 in vitro, and to explore its possible mechanism. Methods： The recombinant adenoviral vector encoding human LASS2 gene was constructed, then the HCCLM3 cells were infected with this recombinant adenoviral vector. The expression of LASS2 protein in HCCLM3 cells was verified by Western blotting. The abilities of invasion and migration of HCCLM3 cells with LASS2 overexpression were detected by Transwell invasion assay and wound-healing assay, respectively. The co-immunoprecipitation was used to verify the junction of LASS2 protein and the subunit of vacuolar H＋-ATPase （ATP6L） in （Co-IP） HCCLM3 cells. The intracellular and extracelluar H＋ concentration of HCCLM3 cells were detected to explore the inhibitory effect of LASS2 overexpression on the function of vacuolar H＋-ATPase. Results： The recombinant adenoviral vector encoding human LASS2 gene was successfully constructed, and the expression of LASS2 gene in HCCLM3 cells was detectable. The abilities of migration and invasion of HCCLM3 cells induced by the overexpression of LASS2 were significantly weakened （P〈0.01）. The invasion ability was decreased to （48.3±7.6）%, and the average number of migrating cells was decreased from 59.00±6.87 to 10.83±3.75. Co-IP provided evidence that the LASS2 protein directly interacted with ATP6L protein in HCCLM3 cells. The change of intracelluar and extracellular H＋ concentration in HCCLM3 cells induced by LASS2 overexpression indicated that the activity of vacuolar H＋-ATPase was suppressed. Conclusion：The overexpression of LASS2 gene in HCCLM3 cells can inhibit the invasion and migration of tumor cells via interacting with ATP6L protein by decreasing the secretion of H＋.