RNA结合基序蛋白8A在肝癌阿帕替尼耐药中的作用及机制研究

阿帕替尼是多靶点肝癌分子靶向药，耐药是限制疗效的瓶颈问题。组蛋白巴豆酰化是新兴的表观遗传调控机制，然而其在肝癌的功能尚不明晰。我们前期报道RBM8A促进肝癌进展，采集阿帕替尼耐药肝癌患者临床样本进行单细胞测序并结合预实验发现：RBM8A促进阿帕替尼耐药，KAT5作为调控其表达的上游分子，可催化组蛋白巴豆酰化激活其转录，进而上调E2F1 mRNA。据此提出：KAT5介导组蛋白巴豆酰化转录激活RBM8A，RBM8A结合E2F1 mRNA进行转录后调控，参与肝癌阿帕替尼耐药。本项目拟运用ChIP、双荧光素酶报告基因、RIP和RNA pulldown等方法，结合巴豆酰活化剂、转录抑制剂等从临床样本、细胞和动物模型多层次解析KAT5催化RBM8A启动子组蛋白巴豆酰化具体位点及协同效应分子，阐明活化的RBM8A与E2F1在转录后层面的互作调控，将揭示RBM8A在肝癌阿帕替尼耐药中的作用及上下游机制。

Apatinib is a small molecular targeting drug inhibiting multiple kinase for treatment of advanced hepatocellular carcinoma (HCC), whereas drug resistance is the bottleneck problem that hinders its therapeutic efficacy in clinical practice. Histone crotonylation is a novel epigenetic regulatory mechanism, but its biological functions in HCC remains unclear. In our previous studies we demonstrated that RNA binding protein 8A (RBM8A) promoted HCC tumorigenesis and progression in vitro and in vivo. Next, we performed single cell sequencing for clinical apatinib-resistant HCC samples and conducted preliminary experiments. We found that RBM8A potentially promotes HCC cells survival and resistant to aptinib. Besides, we found that KAT5 is an upstream regulator of RBM8A, which could promote RBM8A transcription via histone crotonylation activity and thereby increased E2F1 mRNA. Consistent with these, we propose that KAT5 activate RBM8A transcription via a histone crotonylation mediation and subsequently promote RBM8A post-transcriptional positively regulating E2F1 to promote HCC cells resistant to Apatinib. In this study, we will use crotonyl activator, transcription inhibitor, ChIP, dual luciferase reporter gene and performed RIP, RNA pulldown experiments in vitro and in vivo, with the addition of clinical samples validation to explore the specific histone crotonylation sites on RBM8A promoter catalyzed by KAT5 and related synergistic proteins, and investigate the interaction of E2F1 and RBM8A by a post-transcriptional regulating. In summary, our study could potentially reveal a novel specific molecular function and mechanism of RBM8A and its signaling to reverse HCC cells resistant to Apatinib.