miRNA-19a, 19b, 26b在以ET-1和thrombin誘導的CTGF表現及纖維母細胞分化中所扮演的角色

Contribution of miR-19a, 19b, 26b to ET-1 and Thrombin–induced CTGF expression and fibroblast differentiation

林欣樺(Shin-Hua Lin)

指導教授 ： 陳彥州

臺北醫學大學/醫學院/醫學科學研究所/碩士(2013年)

摘要

肺部纖維化 (Pulmonary Fibrosis)是一種嚴重且癒後差的肺部疾病，主要病理特徵是呼吸肺泡上皮細胞之間形成纖維化細胞的集中區，纖維化區主要是由纖維母細胞 (fibroblasts)與分化後的肌纖維母細胞 (myofibroblasts)所組成。Connective Tissue Growth Factor (CTGF)為CCN (Cry61/CEF-10,CTGF/Fisp-12, Nov)蛋白質家族成員，過去發現CTGF大量表現與肺纖維化形成有關，在Thrombin和Endothelin-1(ET-1)處理下會造成肺部大量表現CTGF。MicroRNAs (miRNAs)為一群小分子RNA，參與多種細胞的調節以及人類疾病的發生，其作用機轉為結合到Target genes之3’-UTR進而造成mRNA降解，miRNA表現不正常已被證實與人類疾病發生有密切關係，然而在纖維化過程中，miRNA如何調控CTGF表現目前還不清楚。我們設計了一連串的實驗去探討在in vitro system中miRs在以ET-1與Thrombin誘導肺部纖維化情況下，CTGF表現與纖維母細胞分化的過程中所扮演的角色。實驗結果顯示: 在WI-38細胞中，CTGF蛋白質表現量會被Thrombin和ET-1所誘導，且伴隨著α-smooth muscle actin (α-SMA)以及vimentin蛋白質表現量增加。Thrombin以及ET-1會透過活化mitogen-activated protein kinases (MAPKs)，使得phosphorylated extracellular-signal-regulated kinases (pERK)、phosphorylated c-jun N terminal kinase (pJNK)和phosphorylated p38 mitogen-activated protein kinase (pp38)蛋白質表現量提升而誘導CTGF蛋白質表現;而若另外添加了MAPK的抑制劑則會降低α-SMA以及vimentin的表現。經由miRNA target database (TargetScan, MicroCosm Target, mirbase)分析顯示miRNA-19a，miRNA-19b以及miRNA-26b與CTGF 3’-UTR有結合能力。透過RT-PCR發現在以Thrombin和ET-1刺激之下， miR-19a, miR-19b 和miR-26b的表現量都會隨之下降。而在細胞中大量表現miR-19a, miR-19b 和miR-26b也可以降低Thrombin和ET-1誘導CTGF蛋白表現及纖維母細胞分化的情形。此外，也使用了MAPKs抑制劑以及天然的化合物，包括: quercetin和curcumin，發現它們和miRs一樣也能夠去降低ET-1和Thrombin誘導CTGF蛋白表現及纖維母細胞分化的情形。這些情況都說明了miR-19a, miR-19b 和miR-26b在肺纖維化中扮演著重要的角色，提高miR-19a, miR-19b 和miR-26b的表現能夠保護肺部，使之不走向纖維化的發展。

關鍵字

肺纖維化 ； 結締組織生長因子

並列摘要

Fibrosis is an important cause of morbidity and mortality in the Chronic obstructive pulmonary disease (COPD) patients, and CTGF, which belongs to the highly conserved acronym of Cyr61/CEF-10, CTGF/Fisp-12, Nov (CCN) family of immediate early genes, is a potential target for intervention of lung fibrosis. MicroRNAs (miRs) are small cellular RNAs, and have been implicated in the regulation of a variety of cellular processes, and involved in the pathogenesis of many human diseases. However, the role of miRs in regulation of CTGF expression leading to lung fibrosis is still undefined. In the present study, we designed a series of experiments to examine the role of miRs in regulation of CTGF expression and myofibroblast formation by an in vitro system using a pulmonary fibroblast WI-38 under thrombin or endothelin-1 (ET-1) stimulation.
The results indicated that CTGF protein was induced by thrombin and ET-1 stimulation in WI-38 cells accompanied by increases in ??-SMA and vimentin expression. Activation of MAPKs characterized by increased pERK, pJNK, and pp38 protein expression was observed, and thrombin- or ET-1-induced CTGF, ??-SMA, and vimentin were ameliorated by adding respective MAPK inhibitors. Bioinformatic analysis showed that miR-19a, miR-19b and miR-26b possessed ability to bind CTGF 3’UTR, decreased miR-26b, miR-19a and miR-19b expression by thrombin and ET-1 were examined by RT-PCR. Overexpression of miR-19a, miR-19b and miR-26b significantly reduced CTGF protein expression and myofibroblast formation elicited by ET-1 and thrombin. Furthermore, effects curcumin of MAPKs inhibitors and natural compounds including quercetin and on miRs expression correlated to their inhibitory activities against ET-1 or thrombin-induced CTGF and fibroblast differentiation were investigated. It suggests that miRs including miR-19a, miR-19b and miR-26b play critical roles in pulmonary fibrosis, which increase their expression of miR-19a, miR-19b and miR-26b potentially useful for treating lung fibrosis.

並列關鍵字

lung fibrosis ； connective tissue growth factor ； miRNA

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