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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 homo-sapiens longevity assurance homologue 2 of yeast LAG1 (LASS2) on the metastasis of human hepatocellular carcinoma cell line HCCLM3 in vitro and its possible mechanism. Methods: The recombinant adenovirus containing human LASS2 gene was constructed and transfected into HCCLM3 cells. Western blotting was used to detect the expression of LASS2 protein in HCCLM3 cells. Scratch and in vitro invasion assays were performed to detect the expression of LASS2 gene in HCCLM3 cells. Invasion and metastasis. The co-localization of LASS2 protein and ATP6L in the vacuolar H + -ATPase proton pump was verified by co-immunoprecipitation. Through the determination of intracellular H + concentration , To explore LASS2 overexpression of V-ATPase function inhibition. Results: LASS2 gene was successfully constructed and LASS2 gene was successfully expressed in HCCLM3 cells. LASS2 overexpression significantly decreased the invasion and migration ability of HCCLM3 cells (P <0.01) (48.3 ± 7.6)%, while the numbers of scratched cells decreased from (59.00 ± 6.87) to (10.83 ± 3.75) in the control group; LASS2 protein and ATP6L protein specifically bound in HCCLM3 cells; LASS2 overexpression After intracellular and extracellular H concentration changes significantly, indicating that the V-ATPase proton pump was inhibited. CONCLUSION: LASS2 gene overexpression in HCCLM3 cells can inhibit the migration and invasion of hepatocellular carcinoma cells. The mechanism may be related to LASS2 protein binding to ATP6L and inhibiting its secretion of H +.