The Roles of Micrornas in Endothelin-1 and Hypoxia-Induced Ctgf Production

Fibrosis is an important cause of morbidity and mortality in the lung, and the resident fibroblast has been reported as the primary cells in causing pulmonary fibrosis, however recent findings support that a circulating cell (fibrocyte) also contributes to pulmonary fibrosis. Fibrocytes are bone-marrow-derived mesenchymal progenitor cells, and capable of differentiating into fibroblasts and myofibroblasts, critical cells in pathogenesis of pulmonary fibrosis. A class of small cellular RNAs, termed microRNAs (miRNAs), acting as agents of the RNA interference pathway, can lead to silencing of their relative target genes. Additionally, miRNAs have been implicated in the regulation of a variety of cellular processes, and even the pathogenesis process of human diseases. 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, and recent studies indicated that CTGF is indispensable for persistent lung fibrosis. Data from our team showed that hypoxia and endothelin-1 (ET-1) induced CTGF expression in lung fibroblasts. In cardiac fibrosis, decreases in miRNA-133 and -30 expression have been shown to stimulate CTGF gene egression. In bleomycin-induced mice lung fibrosis, egression of miRNA-155 was elevated, however that was reduced by TGF- stimulation. We further identify miRNA-155 reserves ability to bind with CTGF 3，-UTR using miRNA target databases (TargetScan, MicroCosm Target, mirbase). Although some miRNAs have been implicated in regulating CTGF expression, the critical role played by the expressed miRNAs in regulation of CTGF expression in lung fibrosis is still unclear. In the present project, we will investigate the roles of miRNAs in hypoxia and ET-1-inudced CTGF expression and lung fibrosis in vitro and in vivo. Specific Aim 1 (1st and 2nd year): To define the role of miRNA-155 in ET-1 and hypoxia-induced CTGF expression Hypothesis 1: miRNA-155 participates in ET-1 and hypoxia-induced CTGF expression 1.1. To identify effects of ET-1 and hypoxia on the expression of miRNA-155 in epithelial cells, fibroblast, and fibrocytes. 1.2. To study the causative role of miR-155 in CTGF expression in ET-1 and hypoxia-treated cells. 1.3. To study the regulatory pathway between miR-155 and CTGF expression. 1.4. To investigate the alternative expressions of miRNA-133, -30, 18a, and -17-92 in ET-1 and hypoxia-treated cells. 1.5. To study the signal cascade in ET-1 and hypoxia-induced miRNAs expression 1.6. To study the alternative expressions of miRNA-155, -133, -30, and -17-92 in fibrocytes from normal, mild, and chronic asthma patients (Collaboration with sub-project 1) Specific Aim 2 (1st and 2nd year): To investigate new CTGF-related miRNAs under ET-1 and hypoxia stimulation. Hypothesis 2: CTGF-related miRNAs regulates expression of CTGF under ET-1 and hypoxia stimulation 2.1. MiRNA microarray analysis on control and ET-1 or hypoxia-treated cells to find out new miRNAs which is highly correlated to CTGF expression. 2.2. Analysis of miRNAs expression in cells by quantitative RT-PCR. 2.3. Analysis of the binding capacity of miRNAs with CTGF 35UTR using miRNA target databases (TargetScan, Microsom, mirbase) 2.4. To study the regulatory pathway between miRNAs and CTGF expression elicited by ET-1 and hypoxia 2.5. To examine the expression profile of CTGF-related miRNAs in fibrocytes from normal, mild, and chronic asthmas patients (Collaboration with sub-project 1) Specific Aim 3 (3rd year): To confirm miRNAs expression in bleomycin-induced mouse model of lung fibrosis Hypothesis 3: Alternative miRNAs expression in a mouse model of lung fibrosis 3.1. To examine the ET-1 and CTGF expression in bleomycin-sensitive C57BL/6 and -resistant BALB/c mice 3.2. MiRNA microarray analysis on miRNAs expression profile in the lung tissues from C57BL/6 and BALB/c mice with or without bleomycin stimulation. 3.3. To examine the mechanism of CTGF-related miRNAs egression elicited by belomycin in mice 3.4. To study the in vivo role of miRNAs in lung fibrosis and CTGF egression using miRNAs knockdown probes