肝细胞癌相关乙肝病毒突变体的构建及功能研究

Hepatocellular carcinoma (HCC) is a common malignance in China. It is estimated that 80-90% of HCC is caused by chronic hepatitis B virus (HBV) infection in our country. HBx is a secreted regulatory protein with multi-functions. It is reported to participate the malignant transformation of hepatic cells, especially those with COOH terminal truncated ones. Epidemiological studies with large sample size have discovered several mutations in the X region are significantly associated with an increased risk of HCC. However, which mutations are key factors that promote the occurrence of HCC and their biological functions remain largely unknown. Based on these, in this project, we selected C1653T, T1753C, and A1762T/G1764A, locating in the C terminal region of X gene, whose associations with HCC risk have been repeatedly confirmed and the mutation frequency gradually increased with the progression of asymptomatic HBV carriers to HCC, to study their biological functions. We intended to construct recombinant plasmids containing either wild-type HBx or mutated HBx (single mutation or mutation combinations) through direct gene synthesis or site-directed mutagenesis, respectively. We will compare the protein binding affinity, cell growth, proliferation, invasion, migration, cell cycles, and apoptosis of the cells transfected with different plasmids to clarify the biological functions of these mutations. Then the function classified PCR arrays will be employed to discover the target genes and signal pathways involved in the mutation-related carcinogenic mechanisms.

原发性肝细胞癌是我国常见的恶性肿瘤之一，我国约80-90%的肝细胞癌由乙肝病毒慢性感染导致。HBx蛋白是病毒分泌的一种多功能调节蛋白，被认为和肝细胞的恶性转化密切相关。流行病学研究已发现了一系列HBx区的突变可显著增加肝癌的发生风险，然而对于哪些病毒突变才是肝癌发生过程中的关键突变及这些突变致癌的生物学机制尚不清楚。因此，本课题选择了位于X基因转录激活区、经多项研究证实可增加肝癌发生风险且从携带者发展为肝癌进程中突变频率逐渐升高的突变位点（C1653T、T1753C及A1762T/G1764A）展开功能研究，拟通过体外构建野生型及含单个突变或各种联合突变的HBx重组质粒，观察两者在蛋白结合能力、调节细胞的生长、增殖、侵袭、迁移、细胞周期及细胞凋亡方面的差异，筛选出和肝细胞恶性表型相关的病毒突变或突变组合，并通过功能分类PCR芯片找到突变影响的靶基因和信号通路，探索乙肝病毒突变的致癌机制。

目的 探索乙型肝炎病毒(hepatitis B virus, HBV) X基因(HBx)羧基末端四个突变位点C1653T、T1753C及A1762T/G1764A致癌的生物学机制。方法 利用体外基因合成和定点突变构建野生型和复合突变型HBx重组质粒，转染HepG2细胞筛选稳定表达细胞株。通过绘制细胞生长曲线（CCK8法）、平板克隆形成实验、划痕实验、Transwell侵袭实验比较转染空载体(Vector)、野生型HBx(WT)和突变型HBx(Combo)细胞的增殖、迁移和侵袭能力。结果 细胞生长曲线结果提示WT和Combo细胞的生长速率明显快于Vector（P<0.01）, 但WT和Combo之间无显著差异。平板克隆形成实验结果提示Combo细胞的克隆形成率(31.90%)显著高于WT(16.00%)（P<0.01），而WT略高于Vector(12.46%)（P<0.05）。划痕实验结果显示Combo细胞48h迁移距离显著高于WT细胞（P<0.01），而WT细胞显著高于Vector细胞（P<0.01）。Transwell侵袭实验结果显示Combo细胞穿过小室的细胞数最多，其次为WT细胞，Vector细胞最少，三者相比均具有显著差异（P<0.05）。结论 野生型和突变型HBx均可显著增强HepG2细胞的增殖、迁移和侵袭能力，且突变型HBx促进细胞恶性生物学行为的能力显著强于野生型HBx。