NFAT2沉默机制及对肝癌细胞凋亡影响

The dysfunctional balance between proliferation and apoptosis in hepatocyte is the main principal for genesis of HCC. There is extracellular or intracellular apoptosis pathway activation during HCC formation and development. Our previous work found NFAT family members was silenced in HCC tissues or cell lines and NFAT2 was most remarkable. Restoring NFAT2 expression induced HCC cells apoptosis ,accompanied with MAT1A and FasL up-regulation while Bcl-XL down-regulation. We infer that due to genetic or epigenetic change in HCC，down-regulated NFAT2 suppress its target gene MAT1A transcriptional level，resulting intracellular SAM level falling down，directly or indirectly influencing two important apoptosis-related gene Fasl and Bcl-XL expression ，ultimately causing extracellular or intracellular apoptosis pathway suppressed which is beneficial for HCC cells survival. To verify this hypothesis，first, we will study the NFAT2 silencing mechanism in genetic and epigenetic level.Second, we will study the mechansim of NFAT2 inducing HCC apoptosis in vitro and in vivo,that is whether NFAT2 induce apoptosis by regualting MAT1A expression and SAM level or by directly reguating apoptosis related gene Fasl and Bcl-XL which is following activation of excelluar or intracellur apoptosis pathway.

肝细胞增殖和凋亡之间平衡的失控是肝癌发生的首要条件，肝癌发生发展过程伴随着凋亡内外通路的失活。在我们前期的工作发现NFAT家族在肝癌组织及细胞中是沉默的，其中NFAT2差异最大。恢复NFAT2表达能促进肝癌细胞的凋亡，伴随MAT1A、FasL的表达上调，而Bcl-XL下调。我们推测肝癌中由于遗传或表观遗传上的改变，NFAT2表达降低，抑制了下游靶基因MAT1A转录水平，导致胞内SAM的下降，直接或间接影响FasL和Bcl-XL这两个重要凋亡基因的表达，抑制了细胞内外凋亡通路并有利于肿瘤细胞生存。为了印证这一推测，首先我们将从遗传及表观遗传水平探讨NFAT2沉默的机制。其次我们将从体内体外实验研究NFAT2促肝癌凋亡机制，即NFAT2是否通过调控MAT1A表达及SAM水平来诱导凋亡。或者NFAT2能否通过直接调控凋亡相关基因FasL和Bcl-XL来激活内外凋亡通路而发挥促凋亡作用。

NFAT2被发现与乳腺癌、结直肠癌等多种肿瘤的发生发展有密切相关性，目前与肝癌的相关性尚无文献报道。我们通过免疫组化、Western blot和qRT-PCR发现NFAT2蛋白和mRNA均在在肝癌组织和细胞中低表达。进一步甲基化分析发现NFAT2启动子在肝癌组织中处于高甲基化状态，并且甲基化水平与mRNA成负相关。肝癌组织中NFAT2蛋白与肿瘤直径及AFP水平密切相关。为了了解NFAT2在肝癌中的功能，我们构建了NFAT2过表达载体并将其转入肝癌细胞中，通过CCK-8、细胞克隆形成、细胞周期分析和凋亡分析等方法发现NFAT2能抑制肝癌细胞的增殖和促进肝癌细胞的凋亡。我们进一步通过qRT-PCR筛选下游基因，发现过表达NFAT2能上调FasL的mRNA和蛋白表达。通过CHIP实验进一步证实NFAT2能结合FasL启动子来发挥其调控作用。Western Blot实验发现过表达NFAT2能上调凋亡相关蛋白caspase8和caspase3表达，提示NFAT2可能是通过激活外凋亡通路来发挥其促凋亡作用。本项目首次发现NFAT2在肝癌细胞中的抑制增殖和促凋亡功能，为探讨肝癌疾病的发生发展的机制和今后肝癌靶向药物的治疗奠定了一定的基础。