环状RNA-58055调控miR-181a-5p在黄芩素逆转肝癌细胞株耐药中的作用及分子机制研究

The main factor severely hampered the effects of chemotherapy drugs on hepatoma is the drug resistance. Our previous study found baicalein could reverse the 5-FU resistance of drug–resistant hepatoma cells. The circRNA-58055, a circRNA, was highly suppressed in hepatoma cells after treatment of baicalein using circRNA microarray analysis, but the mechanisms remain unclear. To prove the hypothesis of “baicalein → downregulation of circRNA-58055 → upregulation of target miRNA miR-181a-5p → downregulation of target gene STAT3 → reversing drug-resistance of hepatoma cells”, we will investigate the proliferation, apoptosis, cell cycle, tumor formation in nude mice and concentration of intracellular 5-FU of Bel7402/5-FU cells that over express and knock down circRNA-58055, and examine the expression of circRNA-58055 in drug-resistant hepatoma clinical samples and its relationship with clinicopathological characteristics. To illustrate the molecular mechanism, we will also analyze the effect of expression change of circRNA-58055 regulating miR-181a-5p and STAT3 on reversing drug-resistance of hepatoma cells through using Biotinylated miRNA pulldown-qRT-PCR assay and dual-luciferase reporter gene system, etc. This project will provide new clues and new ideas for revealing the molecular mechanism of reversing drug-resistance in hepatoma cells of baicalein from the perspective of circRNA, and will also provide a theoretical basis of development and application of baicalein.

肝癌对化疗药物产生耐药性严重阻碍其化疗效果。课题组前期研究发现黄芩素可逆转5-FU对肝癌耐药株耐药，且黄芩素处理后耐药株环状RNA（circRNA）表达谱发生改变，我们推测circRNA作为新型调控分子可能参与逆转耐药。本研究通过检测过表达和沉默circRNA-58055后对耐药影响及临床样本中其表达与病理特征的相关性；采用pulldown、荧光素酶报告系统等技术解析58055的靶miRNA miR-181a-5p和下游靶基因STAT3表达变化对黄芩素逆转耐药的影响，证实“黄芩素→下调circRNA-58055表达→上调miR-181a-5p表达→下调STAT3表达→逆转肝癌耐药”之假设，明确circRNA-58055在黄芩素逆转肝癌耐药中的作用及机制。本项目从环状RNA角度探讨黄芩素逆转肝癌耐药作用，为揭示黄芩素逆转肝癌耐药的分子机制提供新思路，并为黄芩素药物的开发和应用提供理论依据。

肝癌对化疗药物产生耐药性严重阻碍其化疗效果，课题组前期发现黄芩素可逆转5-FU对肝癌耐药株耐药，但机制不清。本研究通过环状RNA（circRNA）芯片筛选和qRT-PCR验证、RNase R消化、核质分离、胞内定位、临床样本检测、敲低后对药物敏感性及体内成瘤影响确定目标环状RNA为circ-58055；通过黄芩素干预和敲低circ-58055、Biotinylated miRNA pulldown实验、circRNA与miRNA胞内共定位证实其与miR-181a-5p相互作用；通过考察萤光素酶报告实验、过表达miR-181a-5p、干扰notch2对肝癌耐药株药物敏感性影响、临床样本及细胞株中miR-181a-5p表达特征及与notch2表达相关性、miRNA回复实验考察miR-181a-5p与notch2的关系及对耐药株的影响。结果发现黄芩素通过下调circ-58055上调miR-181a-5p下调靶基因notch2表达，抑制HCC耐药细胞的增殖和克隆形成，阻滞细胞周期于S期，并增加耐药细胞对5-FU、ADM的敏感性。其可能的机制是抑制notch2通路-下调c-Myc-下调CDK2，进而影响细胞周期，阻滞细胞周期于S期，诱导细胞凋亡。项目证实了“黄芩素→下调circ-58055表达→上调miR-181a-5p表达→下调notch2表达→逆转肝癌耐药”之假设，明确了circRNA-58055在黄芩素逆转肝癌耐药中的作用及机制。本项目从环状RNA角度探讨黄芩素逆转肝癌耐药作用，为揭示黄芩素逆转肝癌耐药的分子机制提供新思路，并为黄芩素药物的开发和应用提供理论依据。