U87宿主基因SNHG6通过SETD7调控组蛋白/非组蛋白甲基化在HBV致癌作用中的机制研究

Hepatitis B virus X protein (HBx) plays an important role in carcinogenesis, invasion and metastasis of hepatocellular carcinoma (HCC). Our previous study found that the aberrant expression of small nucleolar RNA (snoRNA) host gene in hepatitis B virus (HBV) related HCC and furthermore the SNORD87 host gene SNHG6 acts as an oncogene. Stable isotope labeling with amino acids in cell culture (SILAC) was used to screen SET Domain Containing Lysine Methyltransferase 7 (SETD7) as the key protein in SNHG6 related carcinogenesis and SETD7 can also regulate the level of STAT1. There are several unresolved issues in this study. Does the HBx/SNHG6/SETD7 pathway regulate histone/non-histone methylation in HBV related carcinogenesis? How does this pathway work? Can SNHG6 promote HBV replication by this pathway? This study aimes to explore the mechanism in the regualtion of HBx/SNHG6/SETD7 pathway on histone/non-histone methylation and HBV replication through Chromatin Immunoprecipitation sequence (CHIP-seq), transcriptome sequence and protein immunoprecipitation by using hepatoma cells, animal model of human disease and clinical tissues.These studies hopes to reveal the role of SNHG6 in the development of HBV related hepatic carcinogenesis and will provide new therapeutic targets for molecular approach to treatment of HCC in the furture.

乙型肝炎病毒X蛋白（HBx）在原发性肝癌的发生、发展以及转移侵袭中均发挥着重要作用。我们前期研究发现乙肝病毒（HBV）相关性肝癌发生发展中小核仁RNA(snoRNA)宿主基因表达失调，其中U87宿主基因SNHG6发挥致癌基因作用。SILAC筛选组蛋白赖氨酸甲基转移酶SETD7作为SNHG6调控通路的关键分子，且可影响STAT1的表达。在这一研究中存在几个尚未解决的问题：HBV致癌机制中是否存在HBx/SNHG6/SETD7通路调控组蛋白/非组蛋白甲基化修饰？这条通路是如何发挥作用的？该通路是否对HBV复制产生影响? 本课题将利用细胞和动物模型及临床样本，采用染色质免疫共沉淀、转录组测序、蛋白免疫共沉淀等技术研究HBx/SNHG6/SETD7通路对组蛋白/非组蛋白甲基化及HBV病毒复制的影响。期望揭示SNHG6在HBV诱发肝癌发生发展中的作用机制，为探求新的分子靶向治疗方法提供理论依据。

原发性肝细胞性肝癌是目前全世界最常见的恶性肿瘤之一，具有调控功能的非编码RNA在表观遗传学水平及转录后水平参与多种恶性肿瘤的发生发展，其中长链非编码RNA是目前研究的热点。SNHG6可以在肝癌中通过抑制SAMe浓度下调基因组的甲基化水平。其分子机制主要是SNHG6可以作为ceRNA竞争性结合miR-1297同时激活两条正反馈环路，即miR-1297/FUS/MAT1A和miR-1297/MAT2A，进而下调MAT1A且上调MAT2A表达。外源性SAMe可以逆转FUS和MAT2A的过表达。通过稳定同位素标记的定量蛋白质组学技术结合生物信息学分析明确SNHG6促进肝癌转移中的两个重要下游分子SETD7 和LZTFL1。SNHG6 通过竞争性结合HNRNPL，抑制RNA 结合蛋白HNRNPL 与SETD7 mRNA 的3'-UTR 区结合，降低SETD7 mRNA 的稳定性，进而通过上调Slug促进肝癌转移。SNHG6 吸附RNA 结合蛋白PTBP1，结合LZTFL1 mRNA 促进其降解，通过影响下游多个EMT 相关基因的表达促进肝癌细胞增殖和转移。通过上述研究，我们明确了SNHG6调控肝细胞肝癌侵袭转移的分子机制，探讨了SNHG6可以作为肝癌细胞基因组低甲基化的扳机点或者分子开关的关键作用。