PIG7和A20的相互作用在白血病发病中的作用研究

PIG7 (p53-induced gene 7) is one of the key genes which involves in the apoptosis induced by p53. In our previous study, It was found that PIG7 was down regulated in leukemia cell. Enforcing the expression of PIG7 could induce the apoptosis and differentiation of leukemia cells and sensitize these cells to chemotherapeutic agents. We had screened a serial of proteins which could interact with PIG7 via yeast two-hybrid systerm. Among them, A20 is a ubiquitin editing enzyme with double function. A20 gene is one of the important controlling gene involving inflammatory reaction and apoptosis. Our initial study indicated that A20 had high level expression in some leukemia cells with low level or without PIG7, while A20 had only low level or not detectable expression in some cell lines accompanying high level PIG7. It indicates that the interact of PIG7 and A20 might participate in the proceeding of leukemia. This project is designed to investigate the binding and the interacting of PIG7 and A20 by two-color fluorescence system. After RNA interference or enforced expressing of A20 and/or PIG7, the emphasis is focus on the effects of the interaction of PIG7 and A20 on the proliferation, differentiation, apoptosis and drug resistance of leukemia cells. The results may help to elucidate the molecular mechanism of the interaction of PIG7 and A20 in the pathogenesis of leukemia. Moreover, the findings may help to discover new targets and provide clinical instruction in the individual therapy of leukemia and the prognosis.

PIG7是p53诱导细胞凋亡过程中涉及的关键基因之一。我们的前期研究发现白血病细胞中PIG7的表达显著下调，过表达PIG7则会促进白血病细胞的分化凋亡，并增强其药物敏感性，既而发现了与PIG7结合的一系列蛋白，其中的A20是一种具有双重功能的泛素编辑酶，参与机体炎症反应和细胞凋亡。我们的初步研究显示在PIG7缺失或低表达的白血病细胞系中A20高表达，而PIG7高表达的细胞中A20缺失或低表达，提示PIG7和A20的相互作用参与了白血病发病过程。本项目拟使用双荧光素标记系统在白血病细胞中研究PIG7与A20的细胞到位及相互作用方式；并通过在白血病细胞中分别沉默或过表达A20和/或PIG7，联合药物处理，研究PIG7和A20的相互作用对白血病细胞增殖、分化、凋亡及耐药的影响，以期阐明PIG7与A20相互作用在白血病中的作用机制，为白血病的个性化干预治疗及预后判断提供新的靶标和临床指导。

PIG7在急性白血病细胞中低表达，并在白血病发生发展中起到重要作用。为了明确其作用机制，我们采用酵母双杂交系统首次鉴定出A20为PIG7的结合蛋白之一。A20亦称为TNF-α诱导的蛋白3（TNFAIP3），是调节细胞生存的重要分子，与多种恶性肿瘤发生发展相关，但在急性白血病(AL)中的生物学功能尚不清楚，因而本项目对A20在AL中的生物学功能及相关分子机制进行了深入研究。研究结果发现A20在多种白血病细胞特别是急性淋巴细胞白血病中高表达，且与PIG7相互作用并定位于细胞浆。成功构建带有A20干扰RNA的慢病毒载体后，经过qRT-PCR和WB检测证实干扰效果明显；在Jurkat、Nalm-6、Reh和Kasumi-1细胞中下调A20表达后，细胞增殖减慢，增殖相关信号分子ERK磷酸化水平显著减低；G0/G1期细胞比例增加，S期比例减低，周期相关信号分子p21和p53表达水平升高。A20下调后Nalm-6细胞在裸鼠中的成瘤能力显著降低。在Jurkat细胞和Reh细胞中，降低A20表达可以明显促进细胞凋亡，且凋亡相关分子BCL-2表达下调。A20表达下调后细胞均对化疗药物柔红霉素或依托泊苷（VP-16）的敏感性增强。而过表达A20则会促进U937细胞增殖，减少细胞凋亡，并降低U937对VP-16的敏感性。本研究结果证实A20可以促进AL细胞增殖、调节细胞周期改变、抑制细胞凋亡、抑制白血病细胞在小鼠体内的成瘤性，并且降低白血病细胞对化疗药物的敏感性，这些生物学功能的变化是通过A20对p-ERK, p53, p21和Bcl-2等信号途径的调节实现的。A20在白血病细胞中的异常表达可能与白血病发生发展及耐药相关，因此针对A20的靶向治疗可能为临床AL的个性化诊疗提供一种新的策略。