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人源性长寿保障基因2 通过与V-ATPase 作用抑制肝癌细胞的生长

唐宁, 金洁, 邓云, 柯荣湖, 沈秋瑾, 樊少华, 覃文新

复旦大学上海医学院，上海 200032； 上海市肿瘤研究所，癌基因及相关基因国家重点实验室，上海 200032；3 江苏 大学生命科学院，镇江 212013

摘要

人源性长寿保障基因2 (Homo sapiens longevity assurance homologue 2, 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。

关键词： 人源性长寿保障基因2 ; 肝癌; 细胞生长; 凋亡; 细胞色素c

分类号：R735.7

[LASS2 interacts with V-ATPase and inhibits cell growth of hepatocellular carcinoma.] [Ariticle in Chinese]

TANG Ning, JIN Jie, DENG Yun, KE Rong-Hu, SHEN Qiu-Jin, FAN Shao-Hua, QIN Wen-Xin

Shanghai Medical College, Fudan University, Shanghai 200032, China； National Laboratory for Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai 200032, China； School of Life Sciences, Jiangsu University, Zhenjiang 212013, China

Abstract

Homo sapiens longevity assurance homologue 2 (LASS2) is a novel gene isolated from a human liver cDNA library by ourlaboratory, and it is a human homologue of the yeast longevity assurance gene LAG1 (Saccharomyces cerevisiae longevity assurancegene). According to our previous results, LASS2 could interact with subunit c of vacuolar type H+-ATPase (V-ATPase), and theoverexpression of LASS2 could inhibit the cell growth of a human hepatocellular carcinoma (HCC) cell line, SMMC-7721. In order tounderstand the role of the interaction between LASS2 and V-ATPase in HCC cell growth, we transiently transfected plasmid pCMVHA2-LASS2 into HCCLM3, a HCC cell line without the significant expression of endogenous LASS2. The pH-sensitive fluorescenceprobes, BCECF and BCECF-AM, were used to measure the intracellular and extracellular H+ concentrations of HCCLM3 cellsrespectively. The effect of LASS2 gene on apoptosis was evaluated with Annexin-V/FITC and propidium iodide (PI) by flow cytometry.Western blot was used to detect cytochrome c (Cyt c) in the cytosol and mitochondria, as well as pro-caspase-3 in cytosol. The resultsshowed that the cell growth of LASS2-transfected HCCLM3 cells was significantly inhibited compared with that of the mock control.LASS2 transfection increased intracellular H+ concentration of HCCLM3 cells, while decreased extracellular H+ concentration. Moreover,LASS2 transfection significantly enhanced the apoptosis of HCCLM3 cells. In LASS2-transfected cells, the amounts of Cyt c increasedin the cytosol, while decreased in the mitochondria. Meanwhile, the expression of pro-caspase-3 in the cytosolic extracts wasdecreased. These results implicate that LASS2 gene might increase intracellular H+ of HCC cells via the interaction with V-ATPase,thereby inducing cell apoptosis through mitochondrial pathway.

Key words: Homo sapiens longevity assurance homologue 2; carcinoma, hepatocellular; cell growth; apoptosis; cytochrome c

收稿日期：2010-02-09　　录用日期：2010-04-28

通讯作者：覃文新　　E-mail: wqin@shsci.org

引用本文：

唐宁, 金洁, 邓云, 柯荣湖, 沈秋瑾, 樊少华, 覃文新. 人源性长寿保障基因2 通过与V-ATPase 作用抑制肝癌细胞的生长[J]. 生理学报 2010; 62 (3): 196-202.

TANG Ning, JIN Jie, DENG Yun, KE Rong-Hu, SHEN Qiu-Jin, FAN Shao-Hua, QIN Wen-Xin. [LASS2 interacts with V-ATPase and inhibits cell growth of hepatocellular carcinoma.] [Ariticle in Chinese] . Acta Physiol Sin 2010; 62 (3): 196-202 (in Chinese with English abstract).