乙肝相关性肝癌肝移植术后肿瘤复发的关键突变基因鉴定及功能研究

Hepatocellular carcinoma (HCC) in our country is closely associated with infection with hepatitis B virus (HBV).Currently, liver transplantation (LT) is the only one which is the most effective radical treatment for HCC patients associated with HBV. However, the therapeutic effect is seriously restricted by the poor prognosis of recurrence of HBV-associated HCC. It has been found that this cancer's initiation and progression resulted from accumulation of genetic changes caused by a variety of factors and interaction of genes. Signaling pathway disorders caused by gene mutations is the first mechanism that plays a key role in HCC metastasis. Thus, we proposed that gene mutations may be the key role of tumor recurrence after LT, and mutant genes and their mutation targets that related with tumor recurrence after LT are very likely to be a new critical factor in prevention of tumor recurrence after LT for HBV-associated HCC. At the present, genetic variation relation with tumor recurrence after LT for HBV-associated HCC is rarely reported. We screened 188 candidate nonsynonymous substitutions related with tumor recurrence after LT by high throughput whole-exome sequencing technologies in our preliminary research. Further，this study will test and verify candidate genes mutations by MassARRAY Genetic Analysis and Sanger sequencing with large samples of HBV-associated HCC patients to obtain candidate mutations of genes which related with recurrence and survival of patients, and further to study the function of these candidate mutations of genes which is able to discover the list of targeted genes which mutations play an important role in HBV-associated HCC. This study may further demonstrate the molecular mechanism of tumor recurrence after LT for HBV-associated HCC and provide a new target for the prevention and treatment of tumor recurrence after LT for HBV-associated HCC patients.

乙肝相关性肝癌（HCC）肝移植术后肿瘤复发导致的不良预后极大制约了肝移植疗效。但是目前肝移植术后肝癌复发的机制尚不清楚。现已发现肝癌具有侵袭、转移的遗传倾向性；肝癌的发生发展是多种因素造成基因突变累积及大量基因相互作用的结果。因此，我们提出基因变异可能是肝移植术后肝癌复发的重要因素，肝移植术后肝癌复发相关的突变基因及其突变靶点极有可能是新的肝癌复发防治关键因素。我们前期工作利用高通量外显子测序技术筛选了乙肝相关性肝癌肝移植后肿瘤复发相关的188个候选突变位点。本研究进一步利用临床大样本采用质谱检测技术 和Sanger测序法对候选突变位点进行双向验证，并分析突变与临床病理学特征及预后的相关性，获得候选突变基因；对其进行肿瘤相关功能研究，从而发现乙肝相关性肝癌肝移植后肿瘤复发的关键突变基因。本研究为揭示乙肝相关性肝癌肝移植术后复发的分子机制奠定基础，为术后肿瘤复发的防治提供新的靶点及理论依据。

肝移植是治疗乙肝相关性肝癌的唯一有效根治性手段，但肝癌肝移植术后肿瘤复发的机制尚不清楚。目前认为基因变异可能是肝癌肝移植术后肿瘤复发的重要因素。本研究利用高通量外显子组测序技术筛选了与乙肝相关性肝癌肝移植后肿瘤复发相关的177个候选突变位点，进一步采用MassARRAY技术和Sanger测序法对177个候选突变位点在164例临床样本上验证，获得了70个SNV及其对应的69个突变基因。采用SNP数据的Logistic回归分析显示TP53，Mybpc2，和MPPE1的MAF大于0.05，但MPPE1突变频率（16.5%）在肝癌复发组织中明显高于原发性肝癌组织及良性肝病组织。单因素统计学分析显示，MPPE1基因突变与TNM分期（P = 0.002）和Child Pugh分级（P = 0.04）有相关性，MPPE1突变肝癌患者术后1 -，2 -和3年的复发率高于未突变肝癌患者（P = 0.02）。Cox多因素分析显示：MPPE1突变是肝癌肝移植术后复发独立危险因素（P = 0.04，HR = 1.969；CI：1.043–3.714），表明，MPPE1突变与肝癌肝移植术后复发有关，可能是肝癌复发风险的预测因子。利用干扰技术沉默MPPE1的功能实验显示：下调MPPE1基因抑制肝癌细胞的增殖、侵袭及迁移能力，诱导细胞周期G1期停滞，促进肝癌细胞凋亡。过表达MPPE1的功能实验显示：上调MPPE1基因促进肝癌细胞的增殖、侵袭及迁移能力，抑制肝癌细胞凋亡。利用RNA测序技术分析显示MPPE1基因可能通过Notch, Wnt/β-catenin and MAPK pathways参与肝癌的发生发展。MPPE1 突变 是肝癌复发的风险因子，可能是乙肝相关性肝癌肝移植术后肿瘤复发防治及预测的新靶点。