ASPP2调控肝癌细胞自噬的研究

Autophagy is an evolutinarily conserved catabolic process in which portions of cytosol and organellles are sequestered into double-membrane vesicle and delivered to the lysosome for bulk degradation. In established tumors (such as liver cancer), autophagy can function as a prosurvival pathway in response to metabolic stresses such as nutrient deprivation, hypoxia, absence of growth factors and the presence of chemotherapy or some targeted therapies that might mediate resistance to anticancer therapies..ASPP2 is the first identified member in ASPP (apoptosis stimulating proteins of p53) family of proteins, which regulate apoptosis through interaction with p53 and its family members. In the previous studies, our group has demonstrated that silence of ASPP2 expression promotes tumor growth and metastasis in hepatocellular carcinoma. But the role of ASPP2 in autophagy in liver cancer is unclear. Our previous results indicates that silence of ASPP2 promotes autophagy in HCC cell lines. Here we investigate the role of ASPP2 in the regulation of autophagy in HCC..First, we will detect the autophagic level by transmission electron microscopy, GFP-LC3 dots analysis, western blotting and RT-PCR after ASPP2 downregulation or upregulation in HCC induced by nutrient deprivation, hypoxia and the presence of chemotherapy. Secondly, we will investigate that how ASPP2, through inhibiting autophagy, suppresses cell proliferation, chemotherapy sensitivity, stemness maintainance of liver cancer stem cells and metastasis via anoikis resistence in HCC. Thirdly, we will investigate the mechanisms of autophagy regulated by ASPP2 in HCC. It will be focused on two aspects. On the one hand, ASPP2 suppresses Beclin1 transcription via regulation with NF-kB/RelA; on the other hand, ASPP2 suppresses the function of PI3KCIII-Beclin1 complex through binding with Beclin1. Finally, the expression of ASPP2 and Beclin1 in 200 HCC tissues will be assessed by immunohistochemistry. The prognostic value of various clinicopathologic factors was evaluated..It is important to understand the mechanisms underlying the involvement of autophagy in liver cancer, which may lead to novel preventive and therapeutic approaches for liver cancer.

自噬是肝癌细胞应对恶劣环境（如血供减少、缺氧和营养缺乏，以及化疗药杀伤作用）的生存机制之一，对肝癌自噬分子机制深入研究将为肝癌诊断和治疗提供新依据。ASPP2是ASPP蛋白家族中的重要成员，是一个新发现的肿瘤抑制因子。我们前期实验显示ASPP2表达量与肝癌发生发展密切相关，但ASPP2对肝癌自噬的作用及其作用机制尚不清楚。本项目研究将利用电子显微镜、免疫荧光和流式检测等技术，从体外细胞生物学实验，体内动物实验以及临床病例分析三个角度，探究ASPP2对肝癌细胞自噬水平的调控，及因此而对肝癌细胞增殖能力，化疗敏感性、肿瘤干细胞干性和失巢凋亡引起的转移能力的影响；探究ASPP2从转录和蛋白结合水平上，调控Beclin1而影响自噬进程的分子机制。在此基础上，进一步加深我们对肝癌发生发展的认识，为肿瘤治疗提供新策略。

原发性肝癌是人类常见的恶性肿瘤，在我国，肝癌死亡率占恶性肿瘤死因的第二位。近年来，大量研究表明自噬与肝癌的发生发展和复发，以及化疗耐药等方面关系密切，因此深入研究自噬与肝癌的相关机制，可以为寻找更加有效的早期诊断和治疗肝癌的靶点提供新的依据。.ASPP (Apoptosis Stimulating Proteins of P53) 蛋白家族是一类调控肿瘤细胞凋亡的蛋白家族，包括ASPP1、ASPP2 和iASPP。ASPP2是ASPP蛋白家族中的重要成员，能与p53结合并特异性增强p53与促凋亡基因启动子的结合，促进细胞凋亡，抑制肿瘤生长。我们前期研究发现ASPP2与肝癌的发生发展有着密切关系，但ASPP2对肿瘤细胞自噬的作用尚不清楚。本课题将针对ASPP2在肝癌细胞自噬中的作用进行深入研究。.首先，我们通过体外细胞实验发现在营养缺乏的环境中，ASPP2下调可以促进肝癌细胞内自噬小体数量的增加，LC3I型向LC3II转化增加，p62降解增强，而自噬抑制后，这种趋势消失，这说明ASPP2下调可以促进肝癌细胞自噬水平的增加。相反，ASPP2过表达可以抑制肝癌细胞的自噬。.然后，我们发现ASPP2是通过调节自噬关键基因Beclin 1而调控细胞的自噬的，作用机制主要有两个方面：.一方面ASPP2可以抑制Beclin 1的转录，这种抑制主要是ASPP2通过与RelA/p65、IkappaBalpha结合，抑制了IkappaBalpha的磷酸化，阻碍了RelA/p65被释放入核，最终抑制了RelA/p65结合到Beclin 1启动子区启动转录，抑制了Beclin 1的表达水平。.另一方面，我们还发现ASPP2可以与Beclin 1蛋白直接结合，抑制Beclin 1与PI3KCIII形成复合物，同时也会抑制Beclin 1与UVRAG结合，最终抑制PI3KCIII酶活性的激活，抑制了自噬小体的形成。.ASPP2通过以上两个机制调控了肝癌细胞的自噬水平。我们也发现ASPP2下调促进肝癌细胞自噬抑制了肝癌细胞的凋亡，增强了细胞在营养缺乏时的存活能力，以及耐受化疗药能力。.本课题阐明了ASPP2在肝癌细胞自噬进程中的作用和机制，对于了解肝癌细胞自噬的调控机制有了新的认识，这将为肝癌细胞的诊断和治疗提供新的策略和新的靶点，为抗肿瘤药物的研发提供新的理论证据。