MiRNA/gene profiling unveils early molecular changes and NRF2 activation in a rat model recapitulating human HCC

Studies on gene and/or microRNA (miRNA) dysregulation in the early stages of hepatocarcinogenesis are hampered by the difficulty of diagnosing early lesions in humans. Experimental models recapitulating human hepatocellular carcinoma (HCC) are then entailed to perform this analysis. We performed miRNA and gene expression profiling to characterize the molecular events involved in the multistep process of hepatocarcinogenesis in the Resistant-Hepatocyte rat model. A high percentage of dysregulated miRNAs/genes in HCC were similarly altered in early preneoplastic lesions positive for the stem/progenitor cell marker cytokeratin-19, indicating that several HCC-associated alterations occur from the very beginning of the carcinogenic process. Our analysis also identified miRNA/gene-target networks aberrantly activated at the initial stage of hepatocarcinogenesis. Activation of the NRF2 pathway and up-regulation of the miR-200 family were among the most prominent changes. The relevance of these alterations in the development of HCC was confirmed by the observation that NRF2 silencing impaired while miR-200a overexpression promoted HCC cell proliferation in vitro. Moreover, T3-induced in vivo inhibition of the NRF2 pathway accompanied the regression of cytokeratin-19 positive nodules, suggesting that activation of this transcription factor contributes to the onset and progression of preneoplastic lesions towards malignancy. The finding that 78% of genes and 57% of dysregulated miRNAs in rat HCC have been previously associated to human HCC as well underlines the translational value of our results. Conclusions: this study indicates that most of the molecular changes found in HCC occur in the very early stages of hepatocarcinogenesis. Among these, the NRF2 pathway plays a relevant role and may represent a new therapeutic target.针对肝癌发生早期阶段基因和/或微小RNA（microRNA，简称miRNA）表达失调的研究，因人类早期病变诊断难度较高而进展受阻。因此，需采用能够模拟人类肝细胞癌（hepatocellular carcinoma，HCC）的实验模型来开展此类研究。本研究针对抵抗性肝细胞（Resistant-Hepatocyte）大鼠模型，通过miRNA与基因表达谱分析，对肝癌发生多阶段进程中的分子事件进行系统表征。肝细胞癌中存在表达失调的miRNA/基因，其在干细胞/祖细胞标志物细胞角蛋白-19（cytokeratin-19，CK19）阳性的早期癌前病变中亦呈现类似的表达变化，这表明多种与肝细胞癌相关的表达失调事件早在癌变进程起始阶段就已发生。本研究的分析还鉴定出在肝癌发生初始阶段异常激活的miRNA/基因靶标调控网络。其中，核因子红细胞2相关因子2（nuclear factor erythroid 2-related factor 2，NRF2）通路激活与miR-200家族上调是最为显著的变化。体外实验证实，沉默NRF2可抑制肝癌细胞增殖，而过表达miR-200a则促进肝癌细胞增殖，这一结果验证了上述表达失调事件在肝细胞癌发生发展中的关键作用。此外，三碘甲状腺原氨酸（triiodothyronine，T3）在体内诱导的NRF2通路抑制，伴随细胞角蛋白-19阳性结节的消退，这提示该转录因子的激活可推动癌前病变起始并向恶性表型进展。本研究中大鼠肝细胞癌内78%的基因与57%的表达失调miRNA，此前已被证实与人类肝细胞癌密切相关，这一发现进一步凸显了本研究结果的转化价值。结论：本研究表明，肝细胞癌中检出的多数分子变化早在肝癌发生的极早期阶段就已出现。其中，NRF2通路发挥着关键调控作用，有望成为肝细胞癌治疗的全新靶点。