关键功能性LncRNA介导微囊藻毒素-LR致肝细胞恶性转化的分子机制研究

Microcystin-LR (MC-LR), the most important toxin in cyanobacteria-polluted water, was reported to be responsible for the increased incidence of hepatocellular carcinoma. However, the mechanism of its carcinogenic action remains undetermined. Results from our preliminary experiment have found that the long non-coding RNA (LncRNA) H19, HULC and HOTAIR displayed up-regulated expression while MEG3 and AFAP1-AS1 were down-regulated in malignant transformed human hepatocyte cell line induced by MC-LR. We speculated that LncRNA may be involved in hepatocarcinogenesis induced by MC-LR. In order to test the hypotheses, MC-LR is initially chosen to establish a malignant transformation model in immortalized hepatocyte cell line. LncRNA expression array analysis was then conducted in the transformed cells, followed by validation of select LncRNA by quantitative real-time reverse transcription PCR, and to define the key functional LncRNA during the cell malignant transformation. Subsequently, the expression of LncRNA in the human hepatocyte cell line is down-regulated by siRNA transfection and up-regulated by cDNA transfection, in order to investigate the role and mechanism of LncRNA in MC-LR-induced cell malignant transformation. Further, the level of MC-LR exposure, LncRNA expression of peripheral blood and hepatocellular carcinoma tissue are measured among people from high- or low-incidence area of Guangxi, in order to judge the value of LncRNA as a biomarker for early diagnosis of MC-LR-related hepatocellular carcinoma and provide a basis for the primary prevention of hepatocellular carcinoma.

微囊藻毒素-LR（MC-LR）是水体水华产生的有毒藻类毒素，流行病学研究表明肝癌的发生与MC-LR污染有关，但其致癌机制未明。申请者预实验发现MC-LR诱导永生化正常人肝细胞发生恶性转化时，长链非编码RNA（LncRNA）H19、HULC及HOTAIR表达上调，MEG3及AFAP1-AS1表达下调，推测LncRNA可能参与MC-LR的致癌效应。本课题拟通过肝细胞恶性转化模型，利用LncRNA芯片筛选并经qRT-PCR技术验证不同转化时期细胞中的差异表达LncRNA，确立影响MC-LR致癌作用的关键功能性LncRNA；采用cDNA转染和siRNA技术正、反向干预上述LncRNA，观察其效应，阐明其作用机制；并检测广西肝癌高/低发地区人群MC-LR暴露水平以及外周血、肝癌组织LncRNA表达水平，判断其作为MC-LR相关肝癌的早期诊断标志物之价值，为肝癌的早期预防和控制提供科学依据。

微囊藻毒素-LR（MC-LR）是水体水华产生的有毒藻类毒素，流行病学研究表明肝癌的发生与MC-LR污染有关，但其致癌机制未明。本课题首先通过建立MC-LR诱导的人肝细胞WRL68的恶性转化模型，动态观察细胞中长链非编码RNA（LncRNA）H19、HOTAIR、HULC、MEG3及AFAP1-AS1表达水平的变化；然后利用慢病毒载体构建LncRNA H19高表达的稳转WRL68细胞株，经MC-LR诱导细胞恶性转化，研究LncRNA H19对MC-LR所致肝细胞恶性转化的影响。最后检测广西地区原发性肝癌患者血清、生活饮用水MC-LR暴露水平以及肝癌组织LncRNA H19、HOTAIR、HULC、MEG3及AFAP1-AS1表达水平。结果发现，MC-LR低剂量长期染毒诱导WRL68细胞发生恶性转化过程中伴随LncRNA H19、HOTAIR、HULC持续高表达，LncRNA MEG3、AFAP1-AS1在细胞恶性转化前期呈高表达状态，在恶性转化期呈低表达状态。LncRNA H19高表达可促进MC-LR诱导的WRL68细胞恶性增殖、锚着独立性生长。地理信息系统（GIS）分析发现，广西原发性肝癌患者血清MC-LR浓度在空间上呈现“南高北低”的分布趋势，肝癌患者血清中MC-LR水平与饮用水中MC-LR水平呈正相关。广西南部的扶绥县原发性肝癌患者的肝癌组织中LncRNA H19、HOTAIR、HULC的表达水平显著高于癌旁组织，而肝癌组织中LncRNA MEG3、AFAP1-AS1的表达水平明显低于癌旁组织。上述结果表明，LncRNA H19在MC-LR所致肝癌过程中可能发挥着重要作用。本结果后续可用于MC-LR的致癌分子机制和早期效应标志的探索，为肝癌的预防和控制提供科学依据。