miR-375在乙肝相关性肝癌的疾病进程及预后中的调控作用

The mortality of hepatocellutar carcinoma (HCC) ranks second among all the tumors in China. And the most important reason of liver cancer is hepatitis B virus (HBV) infection. Furthermore, because of the unobvious symptoms in the early stage, most patients being diagnosed are too late to acquire the right treatment, which result in a low survival rate and poor prognosis. There is still in a lack of serological markers for the early prediction of disease progression and prognosis of HBV-related HCC. Studies have shown that miR-375 may be the specific serological markers for hepatitis B and HCC. However, There is no report on the impact of miR-375 on HBV replication and gene expression, the progression of HBV-related HCC and its clinical symptoms, including the staging and prognosis of HCC. Based on the study of HBV prevention and control of demonstration areas in wuwei, the highest incidence of hepatitis B in China, we will continue to collect the study queues, conduct epidemiological investigation and follow up them to detect the expression level of miR-375 in the serum of the HBV carriers, the patients of chronic hepatitis B infection and HBV-related HCC. Then analyze the correlation between the miR-375 levels and the disease progression, tumor stage, clinical features and prognosis. At the meanwhile, we upregulate the miR-375 expression in the HepG2 cells to observe the effect of miR-375 on HBV transcription, replication and gene expression, as well as on the proliferation, migration and invasion of HepG2 cells, which is going to explore the function and mechanism of miR-375 in hepatitis B-related liver disease progression. The study will further clarify the molecular regulatory networks of occurrence and development in HCC, which can provide important leads for early prediction of disease progression and prognosis of HBV-related HCC and the new drug targets on the treatment of HCC.

肝癌死亡率位居我国肿瘤第2位，HBV感染是我国肝癌发生最主要原因。因其早期症状隐匿，大部分患者诊断时已为中晚期，丧失手术机会，生存率低预后差，目前尚缺乏能对其疾病进程及预后进行准确有效预测的血清学标志物。有研究显示miR-375可能是乙肝及肝癌特异性血清学标志指标，但其对HBV复制与基因表达的影响，与肝癌发病进程、临床症状、分期及预后的相关性均未见报道。本课题在全国乙肝最高发地区乙肝防治示范区现场工作基础上，继续收集研究对象组建队列并开展流行病学调查和随访，检测从乙肝无症状携带者、慢性乙肝感染发展到乙肝相关肝癌进程中血清miR-375表达，分析其与疾病进展、临床特征及预后的相关性；同时在HepG2细胞平台上，通过miR-375增强表达探讨其对HBV复制及基因表达的影响，对肝癌细胞增殖、迁移及侵袭的影响。本研究将进一步阐明肝癌发生发展的分子调控网络，为肝癌的疾病进展及预后的预测提供重要线索。

本项目从宏观人群研究和微观细胞学研究入手，探讨了miR-375表达水平与乙肝相关性肝癌患者疾病进展及临床特征的相关性，以及miR-375对肝癌细胞增殖、迁移和侵袭的影响。主要结果：①健康人群血清miR-375表达量最高，慢性乙肝无肝硬化患者、肝硬化患者和肝癌患者血清miR-375水平依次降低。②血清miR-375表达水平在HBeAg、抗-HBe、HBV DNA阳性患者中显著低于阴性患者。③血清miR-375表达水平与患者肿瘤标志物的相关性分析：血清miR-375在AFP和CEA阳性患者中表达水平均低于阴性患者。④通过诊断试验发现血清miR-375诊断乙肝相关性肝癌患者灵敏度为73.9%，特异度为93.0%，可以作为肝癌的辅助诊断指标。⑤ 乙肝相关性肝癌患者肝癌组织miR-375表达水平显著低于癌旁组织，肿瘤组织miR-375的表达水平与抗-HBc以及HBV DNA滴度呈负相关关系。⑥与HepG2细胞相比，miR-375在 HepG2.2.15细胞中呈低表达。⑦CCK-8实验：细胞上调miR-375表达后的增殖速度小于未转染细胞；转染和未转染的HepG2.2.15细胞的增殖速度均小于转染和未转染的HepG2细胞。⑧划痕实验：与转染细胞相比，未转染的肝癌细胞出现迁移现象。⑨Transwell实验：上调miR-375表达的细胞侵袭数目明显少于未转染细胞；上调miR-375表达的HepG2细胞侵袭能力高于HepG2.2.15细胞。本研究从人群角度首次探讨了从健康人群、乙肝慢性感染、肝硬化到乙肝相关肝癌的转变过程中，血清miR-375表达水平的差异，即随着乙肝相关疾病进程的发展，血清miR-375的表达水平逐渐降低。并且分析miR-375表达水平与HBV感染指标及肝癌各项临床检测指标的相关性，发现血清miR-375表达水平与HBeAg、抗-HBe、HBV DNA、AFP及CEA存在相关性。通过诊断试验评价血清miR-375对乙肝相关性肝癌的诊断价值，结果显示具有较高的灵敏度和特异度，为肝癌的疾病进程的早期预测提供新的重要线索。同时，通过细胞实验，发现HepG2.2.15细胞miR-375的表达水平显著低于HepG2细胞；体外增强miR-375表达，能有效地影响HBV的复制，抑制肝癌细胞增殖、迁移和侵袭能力，发挥着类似抑癌基因的作用。