摘要
LASS2 (homo sapiens longevity assurance homologue 2)是本室从人肝cDNA文库中筛选到的一个与酵母(Saccharomyces cerevisiae)长寿保障基因LAG1 (yeast longevity assurance gene 1)高度同源的人类新基因。LASS2基因定位于染色体lqll,属于LASS蛋白家族中的一员。
在LASS2基因功能的前期研究中,我们发现过表达LASS2可以抑制肝癌细胞SMMC-7721的生长。为了验证这一结果,我们将本室构建保存的pCMV-HA2-LASS2质粒瞬时转染高转移肝癌细胞HCCLM3,未转质粒的HCCLM3细胞作为对照。结果发现外源性LASS2的过表达对HCCLM3细胞生长有抑制作用。为了深入研究LASS2基因调控细胞生长的机理,我们利用流式细胞仪和Western blotting方法分析了细胞的凋亡情况。研究发现,LASS2的过表达可以激活细胞的线粒体凋亡通路,介导细胞发生凋亡。提示LASS2可能通过介导细胞凋亡来发挥其抑制细胞生长的作用。
前期实验还发现,LASS2在高转移肿瘤细胞中低表达,而在低转移肿瘤细胞中高表达；并且,LASS2能与V-ATPase质子泵的c亚基ATP6L相互结合。V-ATPase质子泵是真核细胞膜上存在的一种与H+主动转运有关的跨膜蛋白,其c亚基ATP6L参与H+跨膜通道的形成。目前有很多文献报导V-ATPase质子泵,尤其是c亚基ATP6L与肿瘤侵袭和转移相关。以上提示LASS2可能参与肿瘤细胞的侵袭转移。为了研究LASS2在肿瘤侵袭和转移中的作用,我们利用RNA干扰技术,下调低转移肝癌细胞MHCC97-L中LASS2基因的表达,通过检测V-ATPase质子泵的泌氢功能,对H+敏感的基质蛋白水解酶MMP-2的含量和活性,以及体外侵袭实验,探讨LASS2基因通过调控质子泵的泌氢功能来参与肿瘤细胞侵袭转移的机制。结果显示,当LASS2基因的表达被抑制后,质子泵的泌氢功能增加,细胞外H+浓度升高,细胞外MMP-2的活性形式明显增加,MHCC97-L细胞体外侵袭能力显著增强。提示LASS2基因可能通过调控V-ATPase质子泵参与肿瘤侵袭转移。
综上所述,本研究中我们探讨了LASS2参与肝癌细胞体外侵袭的机制,加深了对LASS2参与肝癌细胞侵袭转移的了解。然而,LASS2调控细胞生长和凋亡的具体途径还不清楚。LASS2具有HOX、TLC两个功能域,它可以通过诱导神经酰胺合成酶活性,增高细胞内神经酰胺含量,导致细胞通过线粒体途径发生凋亡。同时,LASS2还可以通过与V-ATPase质子泵的c亚基ATP6L相互结合,抑制细胞内H+泵出,导致细胞内pH值下降这条通路来引发细胞线粒体途径依赖的凋亡。以上结果为深入研究LASS2在肝癌细胞生长、转移中的作用打下了良好基础。
LASS2 is the novel gene isolated from a human liver cDNA library by our laboratory and it is a human homologue of the yeast longevity assurance gene LAG1 (Saccharomyces cerevisiae longevity assurance gene). LASS2, located on chromosome 1q11, is one of the LASS family proteins.
Overexpression of LASS2 could inhibit the cell growth of SMMC-7721, a human hepatocellular carcinoma (HCC) cell line. In order to understand the role of LASS2 in cell growth, we transiently transfected plasmid pCMV-HA2-LASS2 into HCCLM3, a HCC cell line without the significant expression of endogenous LASS2. The results showed that overexpression of LASS2 could significantly inhibit the cell growth of HCCLM3 cells compared with the mock control. To further conform the inhibitory effect of LASS2 on the cell growth of HCC cells, apoptosis in HCC cells was analyzed by flow cytometry and western blot. The results showed that LASS2 gene could induce apoptosis through mitochondrion pathway. The above data revealed that LASS2 might inhibit cell growth via inducing cell apoptosis.
According to our previous results, the expression level of LASS2 is higher in cancer cells with lowly metastatic potential than those with highly metastatic potential. LASS2 could interact with ATP6L, the 16 kDa subunit of proton pump V-ATPase. Proton pump V-ATPase expresses in plasma membrane that can pump protons into extracellular environment. ATP6L, as one of its subunits, can provide proton a hydrophilic path across the membrane. Recently, many articles have reported that V-ATPase, especially ATP6L, is correlated with the metastatic potential of some cancer cells. These suggest that LASS2 could play an important role in tumorigenesis and metastasis of cancer. To explore the role of LASS2 in cancer metastasis, we used small interfering RNA (siRNA) technology to knock down LASS2 expression in MHCC97-L, a lowly metastatic HCC cell line. Meanwhile, we elucidated the possible mechanisms that LASS2 involved in cancer metastasis by investigating the activity of V-ATPase, the expression and activity of MMP-2 which is pH-sensitive protease, and the in vitro invasion. We found that the proton secretion was increased, the activation of MMP-2 was upregulated and the in vitro invasion was enhanced remarkably after expression of LASS2 was suppressed by siRNA in MHCC97-L cells. These results suggest that LASS2 might involve in cancer metastasis through proton pump V-ATPase.
In conclusion, the knockdown of LASS2 expression could promote the in vitro invasion of MHCC97-L cells. However, the signal transduction that LASS2 is involved in cell proliferation and apoptosis is unclear. LASS2 contains two domains: HOX and TLC, which associate with ceramide synthesis. Thus, LASS2 may induce cell apoptosis through mitochondrion pathway by increasing ceramide. Meanwhile, LASS2 also may trigger cell apoptosis through mitochondrion pathway by which V-ATPase inhibits protons into extracellular environment and causes intracellular acidification. These findings provide a clue to investigate the further mechanisms of LASS2 in cell growth and cancer metastasis.
引文
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