miR-23b/27b/24-1基因簇靶向活化型HSC协同沉默Gremlin1和LOX逆转HF进程

In damage-inflammation-repairation of liver, imbalance of the TGF-β and BMP-7 signaling pathways is a critical step in promoting hepatic stellate cell (HSC) sustained activation. Our study has found high concentrations of TGF-β in inflammation responses upregulates Gremlin1 expression and receives positive feedback from Gremlin1, leading to increased TGF-β expression. Simultaneously, upregulation of Gremlin1 inhibits BMP-7 that could antagonize TGF-β signaling, further intensifying TGF-β signaling activity. Thus, Gremlin1 contributes to impairment of the balance of TGF-β and BMP-7-mediated signal transduction, in turn, accelerating hepatic fibrogenesis. . Binding sites of miR-23b and miR-27b were identified in rat Gremlin1 mRNA 3'-untranslated region (3'-UTR) from positions 3107-3442 nt. miR-23b and miR-27b silence Gremlin1 gene in HSCs respectively and miR-27b also down-regulate the expression of collagen and TGF-β..Lysyl oxidase (LOX) is an extracellular enzyme that catalyzes the oxidative deamination of hydroxylysine and lysine residues in collagen and elastin. The resulting peptidyl aldehyde products spontaneously form covalent cross-links with unmodified lysine residues or with other peptidyl aldehyde residues for formatting of insoluble collagen and elastic fibers as well as mature functional extracellular matrix (ECM). Abnormally increased LOX activity can lead to excessive accumulation of insoluble collagen fibers and is directly associated with fibrotic diseases. The prediction using targetscan online service revealed miR-27b and miR-24-1 potentially bind the 3'-UTR of LOX mRNA.. Therefore, we put forward a scientific hypothesis that miR-23b//27b/24-1 belonging to a gene cluster may synergistically silence Gremlin1 and LOX genes. Silencing Gremlin1 may correct the imbalance of the TGF-β and BMP-7 signaling pathways to inhibit the activation of HSCs and to reduce the secretion of ECM; meanwhile, silencing LOX may block maturation and hardening of ECM. Therefore, the strategy using gene cluster miR-23b/27b/24-1 to silence Gremlin1 and LOX cooperatively may reverse hepatic fibrosis (HF)..As α-SMA is a molecular marker of activated HSCs, its promoter (pSMA), which is a kind of polymeraseⅡpromoter, will be used to direct synthesis of short hairpin RNAs (shRNAs) that mimicked the pre-miRNA structure of the autherntic miR-23b/27b/24-1. This project aims to construct an eukaryotic expression vector pSMP8-miR-23b/27b/24-1-2A-EGFP to achieve the exclusive overexpression of miR-23b/27b/24-1 controled by pSMA8 in activated HSCs, then Gremlin1 and LOX genes can be silenced resulting in reversing HF and the effect can be confirmed through in-vivo or in-vitro studies. In vivo, We will prepare biodegradable CPP-P(NIPAM-co-B15C5Am)-b-PMMA polymeric micelles, which have potassium-ion-responsive characteristics modified the surface by cell penetrating peptides targeting HSCs, to deliver the pSMP8-miR-23b/27b/24-1-2A-EGFP to HSCs.

Gremlin1引起的TGF-β/BMP-7信号失衡，过度激活了肝星状细胞（HSC），分泌大量的细胞外基质（ECM），导致了肝纤维化（HF）；赖氨酰氧化酶（LOX）促进ECM共价交联、硬化，妨碍HF逆转。研究发现，miR-23b和miR-27b能沉默Gremlin1，miR-27b 和miR-24-1能沉默LOX。miRNA-23b/27b/24-1同属一个基因簇，其协同调控所致的Gremlin1和LOX共沉默，有可能矫正TGF-β/BMP-7信号失衡，抑制HSC活化，减少ECM分泌；减弱ECM共价交联、硬化，进而逆转HF。本项目旨在构建含α-SMA启动子和miR-23b/27b/24-1基因簇的真核表达质粒，采用HSC特异性的细胞穿膜肽修饰的钾离子响应型纳米胶束递送，实现仅在活化的HSC中沉默Gremlin1和LOX，通过体内外实验确认miR-23b/27b/24-1基因簇能逆转HF。

肝纤维化肝细胞炎症发展为肝硬化的必经病理过程，它是TGF-β等炎症因子刺激肝星状细胞活化，进而转分化为肌成纤维细胞，导致细胞外基质的合成与降解失衡，过多的细胞外基质沉积于肝脏，形成肝硬化。但是，在临床目前尚无有效的药物抑制该过程。在肝脏发育过程中，miR-23b/27b/24-1基因簇能抑制TGF-β信号，并具有靶向下调多个促肝纤维化基因的潜能，因此，miR-23b/27b/24-1基因簇有可能抑制HSC活化和肝纤维化。在该课题中，慢病毒颗粒递送miR-23b/27b/24-1基因簇感染肝纤维化小鼠后，通过肝脏大体形态、HE染色、Masson染色和Genisis免染色胶原成像，确认了miR-23b/27b/24-1基因簇的表达产物能有效地抑制小鼠的肝纤维化。慢病毒颗粒递送miR-23b/27b/24-1基因簇感染体外大鼠的原代HSC后，通过高通量PCR确认miR-23b/27b/24-1基因簇能有效地下调60个促肝纤维化相关基因的表达。运用Predictor-WJF分析六大类171个肝纤维化基因，结合高通量PCR确认的60个下调的促肝纤维化相关基因，初步确认10促肝纤维化相关的候选基因；通过光素酶报告基因分析法和western blot，确认了miR-23b/27b/24-1基因簇对5个肝纤维化基因（gremlin1、TGFβ2、ITGα2、ITGα5和Lox）具有直接靶向下调作用。通过GO、KEGG和PPI分析后，初步确定5个促肝纤维化靶基因主要通过整合素和TGF-β信号通路进一步下调其他肝纤维化相关基因的表达；并通过western blot验证了miR-23b/27b/24-1基因簇能有效地下调HSC中9个促肝纤维化相关基因（TGF-β2，Gremlin1，LOX，ITGα2，ITGα5，COLIα1，COLIα2，TGF-β1和TIMP1）的表达。因此，miR-23b/27b/24-1基因簇的成员通过直接下调5个促肝纤维化靶基因（gremlin1、TGFβ2、ITGα2、ITGα5和Lox）的表达，继而抑制整合素和TGF-β信号通路，导致HSC的活化抑制，最终引起ECM的分泌减少和降解增加；miR-23b/27b/24-1基因簇的表达产物能有效地抑制小鼠的肝纤维化。该课题将为miR-23b/27b/24-1基因簇应用于肝纤维化和肝硬化的治疗奠定坚实的理论基础。