RBM38结合并促进LncRNA-GAS5稳定逆转肝癌索拉非尼耐药的机制研究

So far sorafenib is the only recommended medicine to treat advanced hepatocellular carcinoma (HCC). However, sorafenib usually provide poor survival benefit for HCC patients due to its drug resistance. RNA binding motif protein 38 (RBM38) is a member of RNA binding protein family (RBPs), plays crucial functions in post-transcriptional control of many important genes. Previous studies have showed that inhibition of RBM38 promoted tumorigenesis and progression in various types of cancers. However, whether RBM38 participates in HCC resistance to sorafenib and its underlying mechanisms remain unclear and need to be further investigated. Our previous studies found that RBM38 was down-regulated in HCC cancerous tissues and tumor progression and resistance to sorafenib were suppressed when RBM38 was up-regulated in HCC cells. Further investigations found that RBM38 could directly bind to LncRNA-GAS5 and prolong its half-life in HCC cells. Meanwhile, inhibition of LncRNA-GAS5 in RBM38-overxpressing sorafenib-resistant HCC cells could regain its resistance to sorafenib. Consistent with these findings, we propose the hypothesis that “RBM38 could reverse HCC resistance to sorafenib via directly binding to LncRNA-GAS5 and promote its stability”. This study is aim to investigate this issue from in vitro, in vivo models and clinical samples to clarify the functions and molecular mechanisms of RBM38 in contributing to sorafenib resistance in HCC, which might discover new potential targets and strategy for reversing sorafenib resistance in HCC.

索拉非尼是晚期肝癌一线治疗药物，耐药是限制其疗效的瓶颈问题。RBM38是一种RNA结合基序蛋白，主要结合RNA发挥转录后调控作用，其失活促进多种肿瘤发展，然而其是否参与肝癌耐药尚不清楚。我们前期工作发现：RBM38在肝癌组织中低表达，将其过表达后显著削弱肝癌的进展并可逆转肝癌细胞索拉非尼耐药；深入研究发现RBM38可直接结合并延长LncRNA-GAS5半衰期，而且在RBM38过表达的耐索拉非尼肝癌细胞中，干扰LncRNA-GAS5亦可回复耐药现象。据此提出了“RBM38结合并促进lncRNA-GAS5稳定，逆转肝癌索拉非尼耐药”的假说。本项目拟通过体外细胞实验与体内动物模型以及肝癌临床样本，多层次深入研究其具体机制。本研究将阐明RBM38与肝癌索拉非尼耐药的潜在关系及作用机制，有望为逆转肝癌耐药提供新的理论依据及潜在治疗靶点。

索拉非尼耐药是限制肝癌疗效的瓶颈问题。RNA结合基序蛋白38(RNA binding motif protein 38, RBM38) 是一种RNA结合基序蛋白，主要结合RNA发挥转录后调控作用，其失活促进多种肿瘤发展，但其是否参与肝癌耐药尚不清楚。我们前期研究发现：RBM38在肝癌组织中低表达，将其过表达后显著削弱肝癌的进展并可逆转肝癌细胞索拉非尼耐药；深入研究发现RBM38可直接结合并延长LncRNA-GAS5半衰期，而且在RBM38过表达的耐索拉非尼肝癌细胞中，干扰LncRNA-GAS5亦可回复耐药现象。据此提出了“RBM38结合并促进lncRNA-GAS5稳定，逆转肝癌索拉非尼耐药”的假说，并通过体外细胞实验与体内动物模型，多层次深入研究其具体机制。首先，构建RBM38低表达及过表达的肝癌细胞索拉非尼耐药模型和裸鼠皮下耐药移植瘤模型，检测RBM38改变对细胞功能、耐药性和移植瘤生长的影响，初步探讨RBM38与肝癌索拉非尼耐药的相关性。发现RBM38在肝癌索拉非尼耐药诱导细胞株中表达降低，过表达RBM38减少体外亲本和耐药肝癌细胞增殖，增加凋亡和细胞周期G1期细胞比例，抑制耐药蛋白表达，降低迁移和侵袭能力，提高肝癌细胞对索拉非尼敏感性。随后，利用RBM38沉默和过表达的肝细胞癌索拉非尼耐药细胞模型建立裸鼠皮下耐药移植瘤模型，发现过表达RBM38抑制索拉非尼耐药肝癌细胞瘤体大小、体积及成瘤速度，并抑制耐药相关蛋白表达，提示RBM38参与肝癌细胞索拉非尼耐药。最后，探讨RBM38通过调控LncRNA-GAS5参与肝癌对索拉非尼耐药的作用机制。分子对接及RIP实验证实RBM38 结合并促进 LncRNA-GAS5 的稳定性。建立LncRNA-GAS5敲低的RBM38过表达索拉非尼耐药肝癌细胞模型，发现在该细胞模型中，肝癌细胞增殖能力增强，凋亡和细胞周期G1期细胞比例减少，耐药蛋白表达升高，迁移和侵袭能力升高，而在裸鼠移植瘤模型中，接种了LncRNA-GAS5敲低的RBM38过表达索拉非尼耐药肝癌细胞的小鼠，瘤体大小、体积、成瘤速度均增加，耐药相关蛋白的表达升高。提示RBM38可通过调控LncRNA-GAS5增加肝癌对索拉非尼的敏感性，从而逆转索拉非尼的耐药性。