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人源性长寿保障基因2(Homo sapiens longevity assurance homologue2,LASS2)是本实验室克隆到的一个与酵母长寿保障基因LAG1高度同源的人类新基因。前期研究显示,LASS2可与V-ATPase质子泵的c亚基ATP6L结合,过表达LASS2可以抑制肝癌细胞SMMC-7721的生长。本研究旨在探讨LASS2对V-ATPase质子泵功能的调节是否参与LASS2对肝癌细胞生长的抑制作用。使用本室构建、保存的pCMV-HA2-LASS2质粒瞬时转染肝癌细胞(HCCLM3),而未转染质粒的HCCLM3细胞作为空白对照,运用CCK-8试剂盒检测细胞生长情况;采用BCECF-AM和BCECF氢离子敏感探针分别测定细胞内、外H+浓度;应用流式细胞仪检测细胞凋亡;Western blot检测线粒体和胞质内细胞色素c(cytochrome c,Cytc)、胞质中的无活性pro-caspase-3的表达情况。结果显示,外源性LASS2的过表达对HCCLM3细胞生长有抑制作用;过表达LASS2使HCCLM3细胞内H+浓度上升,细胞凋亡率提高,线粒体Cyt c释放增加,胞质中pro-caspase-3含量下降。以上结果提示提示LASS2可能通过调控V-ATPase质子泵的功能,提高细胞内H+浓度,从而激活细胞线粒体凋亡途径、抑制肝癌细胞的生长。
Homo sapiens longevity assurance homologue 2 (LASS2) is a human new gene cloned in our laboratory and highly homologous to LAG1, a yeast longevity protection gene. Previous studies showed that LASS2 could bind to ATP6L, a subunit of V-ATPase, and that LASS2 could inhibit the growth of SMMC-7721 cells. This study aimed to investigate whether LASS2 regulates V-ATPase proton pump function in the inhibition of LASS2 on the growth of hepatoma cells. HCCLM3 cells were transiently transfected with the pCMV-HA2-LASS2 plasmids and the untransfected HCCLM3 cells were used as a blank control to detect the cell growth by using CCK-8 kit. BCECF-AM and BCECF hydrogen ion-sensitive probes were used to determine the intracellular and extracellular H concentrations respectively. The apoptosis of cells was detected by flow cytometry. The mitochondrial and cytochrome c (Cytc) and cytosolic inactive pro -caspase-3 expression. The results showed that exogenous LASS2 overexpression had an inhibitory effect on the growth of HCCLM3 cells; over-expression of LASS2 increased the concentration of H + in HCCLM3 cells, increased the apoptosis rate, increased the release of mitochondrial Cyt c, and the pro-caspase-3 content in the cytoplasm decline. These results suggest that LASS2 may be through the regulation of V-ATPase proton pump function, increase intracellular H + concentration, thus activating cell mitochondrial apoptosis pathway, inhibiting the growth of liver cancer cells.