LncRNA GAS5-miR-29b靶向调节TGF-β-Smad3在克罗恩病肠壁纤维化中的作用

Stenosis caused by intestinal fibrosis warrant surgical intervention is the leading cause of the high mortality of Crohn's disease (CD). The dedifferentiation of mesenchymal cells and transformed into myofibroblast (MFB) play a key role in the progression of intestinal fibrosis processes, but the specific mechanism is unknown. Our recent studies found long noncoding RNAs (lncRNAs) GAS5 is highly expressed in MFB in patients with stenotic CD accompanied by microRNA (miRNA)-29b downregulated. miR-29b can inhibit the expression of Smad3 which play a stimulatory role in the pathway of TGF-β- Smads. Thus, whether GAS5 up-regulated Smad3 by targeting miR-29b and whether the downregulation of GAS5 can postpone the intestinal fibrosis processes warrant further study. Based on these findings, By analyzing the differential expression of GAS5 in CD, we will evaluate whether GAS5 can be used as a molecular marker of poor prognosis (fibrotic stenosis) of CD. On the basis of this study, the influence of inflammatory factors on the expression of GAS5 core promoter was studied. Furthermore, the interaction between GAS5 and miR-29b and its possible mechanism will be examined. In addition, We will further test our hypothesis with the use of siRNAs targeting GAS5 both in trinitrobenzesulfonic acid (TNBS)-induced colitis mice models and the murine-derived intestinal organoids, the effect of GAS5 on the progression of chronic TNBS-induced colitis and intestinal fibrosis will be determined. We aim to delineate the mechanism of lncRNA- GAS5-miR-29b in promoting intestinal fibrosis. The anticipated results will provide novel therapeutic approaches for CD with promise to improve the clinical outcomes of CD patients or even reverse intestinal fibrosis.

肠纤维化狭窄致肠道狭窄是克罗恩病（Crohn's disease，CD）高致残率的首因。间质细胞去分化，转化为肌成纤维细胞是肠壁纤维化进程的关键事件。我们前期发现长链非编码RNA(lncRNA)-GAS5在狭窄型CD患者肌成纤维细胞中高表达，同时伴随微小RNA(miRNA)-29b表达下调，而miR-29b可显著抑制TGF-β信号通路刺激性分子Smad3的表达。基于此，我们提出本研究的科学问题：lncRNA GAS5-miR-29b是否通过靶向调控TGF-β-Smads通路参与CD肠壁纤维化？本研究拟在人体组织学、体外细胞学检测lncRNA GAS5在CD中的差异表达，致炎因子对其表达的影响。探讨lncRNA GAS5与miR-29b相互作用及可能的分子调控机制。最后以微小肠及结肠炎小鼠为研究载体，观察siRNA GAS5对肠纤维化的防治效应，为逆转CD肠壁纤维化提供实验基础及潜在靶点。

肠纤维化狭窄致肠道狭窄是克罗恩病（Crohn's disease，CD）高致残率的首因。我们前期发现长链非编码RNA(lncRNA)- GAS5在狭窄型CD患者肌成纤维细胞中高表达，同时伴随微小RNA(miRNA)-29b表达下调。基于此，我们推测lncRNA GAS5 -miR-29b是否通过靶向调控成纤维细胞参与CD肠壁纤维化。本研究拟采用人体组织学标本、体外细胞学、体内动物实验，探讨lncRNA GAS5与miR-29b相互作用及可能的分子调控机制。通过扩大样本量，本研究证实狭窄表型CD患者手术切除肠道标本中lncRNA GAS5的表达上调，并与炎症活动程度相关。通过构建TNBS慢性肠纤维化模型，观察不同时间段GAS5的动态变化，结果显示GAS5在8-12周有显著上升，与炎症及纤维化程度呈正比。通过分离培养原代人成肌纤维细胞，用致炎细胞因子和TLRs激动剂分别刺激上述细胞株，qRT-PCR结果表明，炎症环境下成肌纤维细胞lncRNA GAS5表达的上调。通过送检人成肌纤维细胞RNA sequencing，发现狭窄表型来源的成纤维细胞TGF-β-Smads通路表达明显上调。结合生物信息学分析，TGF-β-Smads可能是lncRNA GAS5作用靶标。通过RNA FISH，我们明确GAS5定位于细胞质，进一步研究表明ceRNA机制可能是其主要效应机制。体内实验表明siRNA GAS5可以减轻TNBS诱导的小鼠肠道纤维化。因此，基于本研究的结果，GAS5可能作为克罗恩病肠道纤维化潜在的治疗靶标。