活化型HSC中MiR-705下调gremlin1表达的调控机制研究

Damage-inflammation-repairation of liver can lead to the activation of hepatic stellate cell (HSC), which trigues TGF-β signal transduction pathway, and then induces hepatic stellate cell to secrete a lot of collagen, causing the disorders of synthesis and degradation of extracellular matrix. This is the key in forming hepatic fibrosis (HF). Our study found that Gremlin1 is the downstream molecule on TGF-β pathway and Gremlin1 was positively correlated to the expression of TGF-β. Gremlin1 can enhance the effect of TGF-β signal pathway through antagonizing BMP7, which accelerats the formation of HF. Therefore, if we take suitable ways to downregulate the expression of Gremlin1 in vivo, the activation of HSC can be prevented or even reversed. Sequence analysis found that 3'noncoding region of the gremlin1 gene settles a structural foundation for negative post-transcriptional control of microRNA. The expression of microRNA-705 is downregulated in activated HSC. Bioinformatics research shows that it can be bound to the 3'UTR of gremlin1 in human, mouse and rat and the overexpression of microRNA-705 has the potential of post-transcriptional suppressing the expression of Gremlin1. As α-SMA is a molecular marker of activated HSC and its promoter (pSMA), which is a kind of polymeraseⅡpromoter (pol-Ⅱ), is going to used to direct synthesis of short hairpin RNAs (shRNAs) that mimicked the pre-miRNA structure of the autherntic rno-miR-705 and will be placed in an intron region downstream of theα-SMA promoter.This project aims to construct Lv-pSMA-miR-705-GFP which belongs to a Lentiviral expression vector expressing pre-miR-705 and is controled by pSMA, and achieve the exclusive overexpression of miR-705 in activated HSC through the characteristic of pSMA being activated exlusively in activated HSC and then trigueing transcription of the pre-miR-705, through which the expression of gremlin1 can be supressed and then the real-time adjustment of TGF-β signal transduction pathway can be achieved, and finally the activation of HSC can be supressed. The effec of dealing with HF can be confirmed through in-vivo or in-vitro studies.

肝损伤-炎症-修复导致肝星状细胞（HSC）活化，启动TGF-β信号转导通路，分泌大量的胶原蛋白，引起细胞外基质的合成与降解失调，是肝纤维化（HF）形成的关键。本课题组研究发现Gremlin1是TGF-β的下游分子，与TGF-β的表达呈正相关，它通过拮抗BMP7，起到强化TGF-β信号的作用，加速HF的形成。若采用恰当的策略在体内下调Gremlin1的表达，有可能起到阻止或逆转HSC活化的作用。序列分析发现，gremlin1基因较长的3′非编码区为miRNA的转录后负调控奠定了结构基础，生物信息学研究显示，在HF中表达下调的miR-705具有抑制Gremlin1表达的潜能，而α-SMA的启动子具有仅在活化的HSC中被激活的特点。本项目旨在通过构建含α-SMA启动子和miR-705的慢病毒表达载体，特异性的实现仅在活化的HSC中下调Gremlin1的表达，通过体内外实验确认其治疗HF的效果。

miRNA在控制发育进程、细胞分化、细胞凋亡以及器官发育等扮演着关键性角色，本课题组前期研究确认，通过siRNA下调Gremlin1的表达可抑制HSC-T6的活化，进而逆转CCL4诱导的SD大鼠肝纤维化模型的纤维化进程。本课题的研究目的主要是筛选并确认HSC-T6中下调Gremlin1表达的miRNA，以及该miRNA抑制HSC活化的能力。本研究通过三组实验解决了该科学问题。首先，采用荧光素酶报告基因分析法，将Gremlin1的mRNA的3′-UTR全长及部分片段所构建的质粒转染HSC-T6，通过荧光素酶的活性筛选下调Gremlin1表达的miRNA的结合区域为G3-23-1（3107-3442 nt）和G3-23-3（3590-3809 nt）。其次，结合生物信息学软件和荧光素酶报告基因分析法，具有与G-3-23-1结合并可能下调Gremlin1表达的保守miRNA有miR-23a/b、miR-27a/b、miR-181a/b/c/d，与G-3-23-3结合并可能下调Gremlin1表达的保守miRNA有miR-182。最后，合成筛选出的miRNA Mimic，将其转染HSC-T6中，通过western blot方法验证miRNA对Gremlin1的下调作用，以及对肝纤维化相关基因（胶原蛋白、TGF-β、α-SMA）表达的影响。结果显示，miR-23b-3p、miR-27b和miR-182能下调HSC-T6中Gremlin1的表达；miR-27b能下调HSC-T6中α-SMA、TGF-β、ColⅠα1和ColⅠα2的表达；miR-181b-1-3p能下调ColⅠα1和ColⅠα2的表达。因此我们认为，miR-27b、miR-23b-3p和miR-182能下调Gremlin1的表达，miR-27b能抑制HSC的活化，这几个miRNA 有可能成为抗肝纤维化的新的分子靶点。