Somatic mutations accumulated in leptin receptor during development of hepatitis C-associated hepatocellular carcinoma

Background & Aims: Hepatocellular carcinoma (HCC) arises in chronic hepatitis and/or cirrhosis via a stepwise accumulation of genetic alterations in various genes. To explore the genetic basis of HCC developed in the hepatitis C virus (HCV)-associated chronic liver disease, we focused on the genetic aberrations latently accumulated in the non-tumorous chronically-inflamed liver. Methods: We determined the whole exome sequences of the 7 paired tumor and the non-tumorous livers. Additional deep sequencing of the representative tumor-related genes and the mutated genes identified were performed on the 22 cirrhotic livers. Functional study of the mutated gene was carried out in vitro and in vivo. Results: Whole exome sequencing clarified that somatic mutations are accumulated in various genes in HCV-infected inflamed liver tissues. Among the mutated genes identified, leptin receptor (LEPR) gene was most recurrently mutated in both tumor and non-tumorous cirrhotic liver tissues. Deep sequencing analyses determined that the LEPR mutations were detectable in 12/22 (54.5%) non-tumorous cirrhotic liver. In vitro downstream validation study demonstrated that the 4/7 mutations determined in the LEPR gene of the cirrhotic livers resulted in the reduction in the levels of signal transducer and activator of transcription 3 phosphorylation, causing the inactivation of LEPR-mediated signaling. Moreover, the LEPR deficient C57BL/KsJ-db/db mice showed higher sensitivity to thioacetamide-mediated liver tumorigenesis compared with control mice. Conclusion: These findings provided the novel evidence showing that somatic mutations are latently accumulated in LEPR in the inflamed liver with chronic HCV infection, providing the putative genesis that enhances the susceptibility for hepatocarcinogenesis.

研究背景与目的：肝细胞癌 (Hepatocellular carcinoma, HCC) 多发生于慢性肝炎和/或肝硬化患者体内，源于多类基因逐步积累的遗传变异。为探究丙型肝炎病毒 (hepatitis C virus, HCV) 相关性慢性肝病继发肝细胞癌的遗传基础，本研究聚焦于非肿瘤性慢性炎症肝脏中潜在积累的遗传异常。

研究方法：本研究对7对配对的肿瘤组织与非肿瘤性肝脏组织完成了全外显子组测序。针对已鉴定出的代表性肿瘤相关基因及突变基因，本研究对22例肝硬化肝脏样本开展了深度测序。此外，本研究在体外 (in vitro) 及体内 (in vivo) 环境中对该突变基因开展了功能验证实验。

研究结果：全外显子组测序结果显示，HCV感染的炎症性肝脏组织中，多种基因均存在体细胞突变的积累。在已鉴定的突变基因中，瘦素受体 (leptin receptor, LEPR) 基因在肿瘤组织与非肿瘤性肝硬化肝脏组织中均为最频繁发生突变的基因。深度测序分析证实，22例非肿瘤性肝硬化肝脏样本中，有12例（54.5%）可检测到LEPR基因突变。体外下游验证实验表明，肝硬化肝脏样本LEPR基因中鉴定出的7个突变位点里，有4个可导致信号转导与转录激活因子3 (signal transducer and activator of transcription 3, STAT3) 的磷酸化水平降低，进而引发LEPR介导的信号通路失活。此外，与对照小鼠相比，LEPR缺陷型C57BL/KsJ-db/db小鼠对硫代乙酰胺诱导的肝肿瘤发生具有更高的易感性。

研究结论：本研究结果提供了全新证据，表明慢性HCV感染所致炎症肝脏中，LEPR基因可潜在积累体细胞突变，这一发现提出了一种可能的发病机制，可增强肝细胞癌变的易感性。