内质网应激相关超级增强子对肝癌细胞自噬的调控及其对化疗药物敏感性的影响

Hepatocellular carcinoma (HCC) is the most common malignant hepatobiliary disease.It is well believed that autophagy induced by endoplasmic reticulum stress plays an important role in chemotherapeutic drug sensitiveness.Currently, super enhancers is becoming a hot topic in cancer area. However, the molecular and cellular mechanisms underlying the relationship between super enhancers and autophagy induced by endoplasmic reticulum stress are largely unknown and still needed further investigation. Our previous studies found that 197 SEs in hepatoma cells significantly changed after ERS stimulation using H3K27ac ChIP-seq and transcriptome sequencing platform.The target genes of these SEs may regulate autophagy induced by endoplasmic reticulum stress through such as IRE1 and PI3K-AKT pathways.Inhibition of SEs by JQ1 can weaken autophagy. Base on these finding, we propose the hypothesis the autophagy mediated by ERS is closely related to the ERS specific super enhancer and its binding transcription factors.To test this hypothesis, we first used methods such as CRISPR/Cas9 and siRNA to perform the functional components and transcription factors of specific SEs on autophagy induced by endoplasmic reticulum stress in drug resistance .Furthermore,the functions of super enhancers including their target genes and related signal pathways will be predicted and confirmed.Lastly, gene editing of hepatoma cells on tumor-bearing mouse models will be performed to test the effects of super enhancers on autophagy induced by endoplasmic reticulum stress and chemotherapeutic drug sensitiveness.The proposed studies above not only could further precisely demonstrate the molecular mechanisms of autophagy induced by endoplasmic reticulum stress but also provide solid evidence and basis for developing novel drug target in the future.

原发性肝癌是常见恶性肿瘤。内质网应激（ERS）介导的自噬是影响肝癌药物敏感性的重要原因。超级增强子（SEs）是目前肿瘤研究热点，但与ERS及自噬的关系未见报道。我们前期利用组蛋白修饰H3K27ac ChIP-seq和转录组测序，发现肝癌细胞中有17个ERS特异性SEs，这些SEs的靶基因可能通过IRE1及PI3K-AKT等通路调节自噬，用JQ1抑制SEs可减弱自噬。由此推测，肝癌细胞ERS介导的自噬与其特异性SEs及结合的转录因子密切相关。本项目拟在前期基础上，通过CRISPR/Cas9和RNA干扰等技术调控ERS特异性SEs的功能元件和相关转录因子，观察肝癌细胞自噬活性变化及对药物敏感性的影响，并阐明相关信号通路。进而制备基因编辑肝癌细胞小鼠模型，在整体水平上研究ERS特异性SEs调控的自噬对药物敏感性的影响。本研究将进一步阐明肝癌细胞ERS介导的自噬机制，并有望发现新型肝癌治疗靶点。

我们利用H3K27acChIP-seq 高通量测序同时联合转录组测序的结果，筛选并鉴定肝癌细胞内质网应激潜在相关的超级增强子，通过生物信息学预测超级增强子的靶基因，进行GO 和KEGG 分析以及GSEA 分析，预测与肝癌细胞凋亡抵抗相关的超级增强子、转录因子及可能的生物学机制，有望发现抑制肝癌凋亡抵抗的新型作用靶点。随后，我们探究了内质网应激相关超级增强子最易结合的转录因子在ZNF263在内质网应激介导的肝癌细胞凋亡抵抗中的潜在作用。我们检测了ZNF263在人类肝癌组织和HCC细胞系中ZNF263的表达。研究了RNA干扰ZNF263对体外HCC细胞增殖、凋亡、药物抵抗性的影响。此外，我们评估了ZNF263敲低对内质网应激的HCC细胞自噬水平及药敏性的影响。说明ZNF263可能参与了超级增强子及其转录因子对肝癌细胞药敏性的调控，为肝细胞癌的治疗提供了新思路。