RBM8A在肝细胞癌中通过EMT调节奥沙利铂耐药的研究

Oxaliplatin(OXA) resistance is urgently needed to be solved in the treatment of hepatocellular carcinoma (HCC). It was found that OXA resistance is associated with epithelial-mesenchymal transition (EMT).Our previous work demonstrated that RBM8A was highly expressed in HCC primary tumors and induced EMT in HCC cell lines. RBM8A (RNA-binding motif protein 8A),as a RNA-binding protein, mainly regulates the clearance of abnormal mRNA and widely participates in tumor progression. However, it still remains unknown that whether the EMT driven by RBM8A plays a role in the development of OXA resistance. The present study aims to: (1) Establish OXA-resistant RBM8A-silencing or RBM8A-overexpressing human HCC cell lines. (2) Evaluate the cancer cell biological functions, drug resistance and EMT characteristics in the HCC cell lines; screen out and testify the differentially-expressed gene by gene microarrays and further determine the downstream pathways. (3) Clarify the mechanism of RBM8A-regulated OXA resistance via EMT in HCC in animal model. (4) Follow-up study is carried out to investigate the correlation of RBM8A expression level with the clinical effect of HCC patients receiving OXA. Our study is going to clarify the potential relationship between RBM8A and OXA resistance in HCC and may offer novel theoretical proofs and potential target for the reversal of drug resistance.

奥沙利铂耐药是肝癌治疗中亟待解决的难题。上皮-间充质转化（EMT）已被证实与奥沙利铂耐药密切相关。我们前期研究发现：RBM8A在肝癌组织中高表达，并且可诱导体外细胞株的 EMT。RBM8A是一种RNA结合基序蛋白，主要调控对异常mRNA的清除，并广泛参与肿瘤发展，但能否通过所诱导的EMT参与奥沙利铂耐药尚不清楚。本研究拟：1)构建RBM8A沉默及过表达的人肝癌细胞模型，建立相应的奥沙利铂耐药株。2)评价各细胞株的肿瘤相关功能、耐药性、EMT特征，基因芯片筛选差异表达基因并进行下游验证。3)动物实验研究RBM8A通过EMT调节奥沙利铂耐药的作用。4)临床随访分析RBM8A的表达水平与接受奥沙利铂化疗患者疗效的相关性。本研究将阐明RBM8A与肝癌奥沙利铂耐药的潜在关系，有望为逆转肝癌耐药提供新的理论依据及潜在治疗靶点。

奥沙利铂(OXA)耐药是肝癌治疗中的巨大障碍，上皮-间充质转化(EMT)已被证实与奥沙利铂耐药密切相关。RNA结合基序蛋白8A(RNA binding motif protein 8a, RBM8A)是外显子连接复合物的核心组件，也是无义介导的mRNA衰变中所必需的蛋白之一，与RNA定位、转录、转运、翻译和降解等有关，还与细胞周期及细胞凋亡调控密切相关，现有研究证实RBM8A广泛参与肿瘤发展。我们前期研究发现：RBM8A在肝癌组织中高表达，与患者不良预后相关；RBM8A高表达可诱导体外细胞EMT，抑制肝细胞癌凋亡，促进其增殖、迁移和侵袭。但RBM8A能否通过诱导EMT参与奥沙利铂耐药尚不清楚。为进一步了解RBM8A在肝癌OXA耐药的作用，阐明RBM8A在肝癌耐药网络中的分子机制，进而探索逆转肝癌耐药的潜在靶点。首先，构建RBM8A低表达及过表达的肝细胞癌OXA耐药模型和裸鼠皮下耐药移植瘤模型，检测RBM8A改变对细胞功能、耐药性和移植瘤生长的影响，初步探讨RBM8A与肝癌OXA耐药的相关性。发现RBM8A在肝癌OXA耐药诱导中表达升高，过表达RBM8A促进体外亲本和耐药肝细胞癌增殖，减少凋亡和细胞周期G1期细胞比例，诱导耐药蛋白表达升高，提高肝细胞癌对OXA耐受性，提示RBM8A参与肝细胞癌OXA耐药。随后，利用RBM8A沉默和过表达的肝细胞癌OXA耐药模型模型和裸鼠移植瘤模型，检测RBM8A改变后细胞骨架、EMT特征和细胞迁移、侵袭的变化，探讨RBM8A通过EMT参与肝癌对OXA耐药的作用机制。发现过表达RBM8A诱导亲本和OXA耐药肝细胞癌骨架呈现间质样细胞表型，抑制上皮细胞标志物E-cadherin的表达，促进间质细胞标志物N-cadherin和Snail的表达，促进细胞迁移和侵袭，RBM8A通过诱导EMT参与肝细胞癌OXA的耐药。最后，通过高通量芯片技术和生物信息学方法，分析RBM8A沉默和过表达的OXA耐药肝细胞癌模型中差异表达基因网络，并结合体外验证实验筛选潜在调控因子和信号通路，发现RBM8A在肝细胞癌对OXA耐药过程中具有广泛的转录调控作用，RBM8A可能通过LncRNA MALAT1、LncRNA FENDRR、STAT3和HDAC9等关键调节因子以及胰岛素信号通路，MAPK信号通路和PI3K-Akt信号通路等诱导EMT，参与肝细胞癌OXA耐药。