CircEphB4抗肝癌新机制及白藜芦醇对其的调控研究

Circular RNA (circRNA) is covalently closed non-coding RNA. As circRNA is stable, abundant, and involved in regulation of a variety of diseases such as cancer, circRNA has been become prospective novel target of cancer therapy. However, the role of circRNA in hepatocellular carcinoma (HCC) cells and the mechanism by which it functions are still unclear. Recently, we found that circEphB4 was downregulated in HCC tissues comparing with non-tumor tissues. Overexpression of circEphB4 inhibited the proliferation and metastasis of HCC cells. Transcriptome sequencing indicated that chemokine CCL20 and its downstream phosphorylated ERK is correspondingly downregulated in circEphB4-overexpressing stable HCC cell lines. Therefore, we hypothesized that circEphB4 may suppress the proliferation and metastasis of HCC cells via CCL20/ERK pathway. Further, we screened out resveratrol, a monomer of Chinese traditional herbs that inhibited the viability of HCC cells and upregulated the expression level of circEphB4. In this study, we will further elucidate how resveratrol induced the upregulation of circEphB4 and the mechanism by which circEphB4 targeted CCL20 for its regulation with high throughput sequencing, RNA-pull down and RNA immunoprecipitation, in vitro and in vivo experiments, and clinical tissue samples. By revealing the upstream and downstream signal pathways through which circEphB4 inhibits the proliferation and metastasis, this investigation will provide scientific evidence for that circEphB4 is identified as a novel target of HCC treatment and drug development.

CircRNA是一种共价成环且结构稳定的新型非编码RNA，其表达丰富且参与肿瘤等多种疾病调控，是癌症治疗潜在新靶点，然而在肝细胞癌中作用和机制不清。近来我们发现肝癌中circEphB4表达显著下调，过表达circEphB4抑制肝癌增殖和转移，转录组测序提示circEphB4下调趋化因子CCL20及其下游ERK磷酸化水平。由此我们提出：circEphB4通过调控CCL20/ERK信号通路抑制肝癌增殖和转移的新机制。进一步，我们筛选到中药单体白藜芦醇抑制肝癌细胞活力并上调circEphB4表达。因此，本项目拟进一步通过高通量测序、RNA-pull down、RNA IP、体内外实验以及临床标本分析，阐明白藜芦醇诱导circEphB4上调及其靶向调控CCL20的分子机制，揭示circEphB4调控肝癌增殖和转移的上下游机制，为确立circEphB4作为肝癌治疗新靶点和针对性药物开发提供充分科学.

本研究主要阐明了circEphB4抑制肝癌细胞增殖和转移的分子机制，并通过动物实验验证了circEphB4抗肿瘤潜力。另外，预实验提示白藜芦醇能够上调肝癌细胞circEphB4，而进一步实验却发现白藜芦醇上调circEphB4并不显著，将circEphB4干扰后，白藜芦醇的抗肿瘤效果也没有符合预期得到逆转。然而，有趣的是白藜芦醇能够上调肝癌细胞缝隙连接蛋白，提示增强肝癌细胞之间细胞通讯。基于此，我们将白藜芦醇联合自杀基因HSK-TK/GCV，发现白藜芦醇显著增强自杀基因对肝癌细胞旁杀伤效应，进一步动物实验也验证了两者的协同杀伤作用。问题是缝隙连接蛋白属于长半衰期蛋白，其降解主要溶酶体自噬途径，那么白藜芦醇能够上调缝隙连接蛋白，提示白藜芦醇或干预自噬，文献报道的确印证上述观点，并且理论上抑制自噬有望增效化疗杀伤肿瘤。受到启发，我们筛选了系列中药小分子，其中常春藤皂苷元，土贝母皂苷能够抑制细胞自噬，正如之前推测，协同化疗的确能够较好杀伤肿瘤细胞。