miR-34c靶向ACSL1促进肝纤维化发生发展的机制研究

Nowadays the incidence of liver fibrosis is increasing quickly but still lack of effective ways for prevention and treatment.The activation of hepatic stellate cells (HSC) and its related adjustment mechanism is considered as the key to hepatic fibrosis development. miRNA and its associated molecular mechanism gradually show its potential value of clinical diagnosis and treatment for chronic liver disease. In our preliminary work ,we discovered that rising miR-34c expression was positively correlated with the development process of the liver fibrosis in DMN-induced rat models, and long-chain acyl coenzyme A synthetase 1 (ACSL1), the miR-34c's target gene, expressed down in this process. But the specific function of both miR-34c and ACSL1 in the process of liver fibrosis has not been clarified yet. Based on these results,we assumed that miR-34c promotes the activation of HSC and thus promotes the development of hepatic fibrosis by targeting ACSL1. Therefore,We plan to ①confirm that the expressions of miR-34c was associated with clinical hepatic fibrosis process by detection of its expressions in clinical liver samples; ②by means of cell functional experiments in vitro:study the biological function of miR-34c in HSC activation/liver fibrosis progression through regulating the expression level of miR-34c in HSC; ③clarify the mechanism: miR-34c promotes the development process of liver fibrosis by targeting ACSL1;④try to delay the process of liver fibrosis in vivo by using miR-34c antagonists to interfere with the animal models. This study will improve our understanding of mechanism of liver fibrosis development, and offer potential new molecular targets and perspectives for the prevention and treatment of liver fibrosis.

肝纤维化发病率日趋升高且缺乏有效防治手段，肝星状细胞（HSC）活化及相关调控机制是其发生发展之关键环节；miRNA及其相关分子调控机制正逐渐展现其在某些慢性肝病中的潜在临床诊治价值。本组前期工作发现:DMN诱导大鼠肝纤维化模型中miR-34c表达与肝纤维化进程呈正相关，其靶基因ACSL1在此进程中下调，但未讨论两者在肝纤维化进程中的具体功能。本项目假设: miR-34c靶向ACSL1调控HSC活化促进肝纤维化进程。拟通过①临床样本检测：明确miR-34c表达与临床肝纤维化进程相关；②细胞实验：调控miR-34c在HSC中表达水平，研究其在HSC活化增殖/肝纤维化进程中的生物学功能；③机制探讨：miR-34c促进肝纤维化进程是经靶向ACSL1而实现；④探索miR-34c拮抗剂干预动物模型以延缓体内肝纤维化进程。实施该项目可加深对肝纤维化发生发展机制的认识，为肝纤维化防治提供新的分子靶标和视角

本课题经HSC功能实验及SD大鼠体内实验证实miR-34c可直接靶向ACSL1调控HSC活化增殖而促进肝纤维化。以前期工作为基础提取和鉴定大鼠原代HSC，检测到miR-34c在其自然活化过程中显著上调；在活化态HSC中成功转染miR-34c inhibitor后，HSC活化增殖能力减弱、α-SMA和COLI合成减少。说明miR-34c可促进HSC活化增殖而影响肝纤维化进程。双荧光报告实验验证ACSL1为miR-34c的直接靶基因;利用病毒载体构建了低表达ACSL1稳定细胞株Y2313,在Y2313细胞中转染miR-34c inhibitor后ACSL1蛋白表达升高,肝纤维化指标α-SMA和COLI表达下降,说明Rescue assay实验miR-34c inhibitor可部分回复ACSL1的作用，进一步验证了miR-34c靶向ACSL1调控HSC活化促进肝纤维化进程。动物实验：予miR-34c agomir(模拟剂)干预SD大鼠，观察到miR-34c可在一定程度上促进体内肝纤维化形成；苏丹黑B染色观察到注射miR-34c模拟物后肝组织脂滴形成明显被抑、影响脂代谢，结果正在组织撰写论文。.另一方面重要结果是：前期芯片筛选肝纤维化进程中差异表达miRNA时，除miR-34a/c外也发现了miR-185、miR-484等在肝纤维化进程中显著变化，因此本基金也资助了同时开展的相关平行实验并发现:miR-185在肝纤维化患者外周血中显著上调，可作为诊断肝纤维化的外周血循环标记物，miR-185靶向SREBF1调控HSC细胞活化增殖进而参与肝纤维化进程，论文已发表在Scientific Reports杂志;也发现MiR-185可靶向SCARB1调控HSC的脂代谢/活化和肝纤维化；还发现miR-484通过靶向HSC中Fis1调控肝纤维化进程，论文发表在第二军医大学学报上；且发现miR-484 通过Wnt/β-catenin 信号通路靶向HIPK1调控肝纤维化进程，SCI论文投稿中。纵观上述研究，我们发现这些差异的miRNA及其靶基因跟脂代谢密切相关，故拟进一步研究脂代谢与肝纤维间的相关机制。.项目资助已发表SCI1篇，核心期刊1篇；培养博士生1名，硕士生2名；项目批准经费65万元，已花费48.8735元，结余16.1265万元，剩余经费将用于本研究相关后续支出。