The Role And Mechanism Of MiR-145in TGF-β/Smad3Profibrotic Pathway In Scleroderma

Background:Scleroderma is an autoimmune disease characterized by general fibrosis, immunological abnormalities and vascular injury. Skin is the most frequently involved organ, with increased accumulation of extracellular matrix proteins in the skin. It often affects various internal organs, such as lung, heart, kidney, and digestive tract, leading to severe complications such as pulmonary interstitial fibrosis and pulmonary hypertension. Ten-year survival rate of scleroderma is60%～90%. Because the etiology of scleroderma has not yet been fully elucidated, there is no specific treatment for scleroderma patients, especially target to fibrosis. A growing body of evidence suggests that extracellular matrix overproduction by activated fibroblasts results from complex interactions among endothelial cells, lymphocytes, macrophages, and fibroblasts via a number of mediators, such as cytokines, chemokines and growth factors. It is crucial to interpret the pathogenesis of scleroderma to understand clinical classification, explore new treatments and judge prognosis.Fibrosis in skin and internal organs is the most important physiopathological process in scleroderma, in which transforming growth factor-β (TGF-β) plays a central role. TGF-β is a strong chemoattractant for fibroblasts. TGF-β increases the synthesis of extracellular matrix, such as collagen type Ⅰ and type Ⅲ, or fibronectin by fibroblasts, modulates cell-matrix adhesion protein receptors, and regulates the production of proteins such as plasminogen activator or procollagenase, which can modify the extracellular matrix by proteolytic action. In addition, TGF-β is capable of stimulating its own synthesis by fibroblasts through autoinduction. TGF-β increases TGF-β receptor levels in fibroblasts, and thus the maintenance of increased TGF-β production my lead to the progressive deposition of extracellular matrix, resulting in fibrosis. TGF-P signaling occurs predominantly by phosphorylation of cytoplasmic mediators belonging to the Smad family, especially Smad3. In the presence of TGF-β ligand, Smad3is phosphorylated directly by the TGF-β receptor I kinase, and bind to Smad4. The resultant complex migrates into the nucleus to interact with the Smad binding element and transcription factor. Then gene expression is inducted, including the elevated expression of genes encoding extracellular matrix.microRNAs (miRNAs) are recently discovered22-nucleotide-long noncoding RNAs, which negatively regulate gene expression by base pairing with the3-untranslated region (UTR) of their target messenger RNAs (mRNA). If pairing is perfect or nearly perfect, target mRNAs are degraded. When their pairing with mRNAs is imperfect, it results in translational repression. In the last years, the number of known miRNAs has grown exponentially, and currently more than1000miRNAs are known to be encoded by the human genome. miRNAs play an important role in the pathogenesis of many autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, Sjogren’s syndrome and primary biliary cirrhosis. The latest studies show miRNAs are likely to participate in the pathogenesis of scleroderma. But the specific miRNAs and their target genes are still needed to be further studied.miRNA microarray technique is a newly developed high-throughput screening method which can detect different expression of miRNAs in patients and healthy controls. This study is aimed to screen differently expressed miRNAs in scleroderma patients and normal controls by miRNA microarray technique, identify miRNAs related to TGF-β/Smad profibrotic pathway through bioinformatics analysis, and testify the role of target miRNA and its target gene in the level of tissue and cell in the pathogenesis of scleroderma by real-time polymerase chain reaction (PCR) and immunohistochemisty. This study will promisingly provide experimental evidence to illuminate the role of miRNA in the pathogenesis of scleroderma, and offer target miRNA to specific anti-fibrotic treatment for scleroderma.Part One Different expression of microRNA in skin of patients with sclerodermaObjective:To compare miRNA expression profile between scleroderma patients and normal controls, and identify differentially expressed miRNAs in patients with scleroderma.Methods:Clinical data of seven patients with scleroderma were collected. Skin samples of scleroderma patients and seven controls were obtained from forearm for pathologic examination, fibroblast culture and miRNA microarray analysis. Differently expressed miRNAs were analysed by miRNA microarray chip technique.Results:In seven patients with scleroderma, five cases were diffuse scleroderma, and two were limited scleroderma. Two patients were male, and five were female. They were30～64years old, with average age of49.0years. The average course of disease was16.6months. In seven controls, four were male, and three were female. They were21～58years old, with average age of44.8years. The biopsy of skin samples of scleroderma patients showed collagen excess deposition and deep fibrosis. miRNA microarray chip analysis identified42miRNAs differently expressed in the diffuse scleroderma skin tissue (including25upregulated miRNAs and17downregulated miRNAs). In addition,60miRNAs appeared to be changed in the limited scleroderma skin samples (including42upregulated miRNAs and18downregulated miRNAs).21miRNAs altered in common both in diffuse and limited scleroderma (including13upregulated miRNAs and8downregulated miRNA).Conclusion:There are different miRNA expression profiles in skin tissue between scleroderma patients and normal controls.Part Two The role and mechanism of miR-145in TGF-β/Smad3pathway in sclerodermaObjective:To investigate the role and mechanism of miR-145in TGF-β/Smad3pathway in sclerodermaMethods:miRNAs related to TGF-β/Smad3pathway were screened by bioinformatics analysis. The expression of miR-145in skin and skin fibroblast of scleroderma and normal control were tested by real-time PCR. Smad3protein and mRNA in skin and skin fibroblasts of scleroderma and normal control were detected by immunohistochemisty and real-time PCR. In the presence of TGF-β, miR-145and Smad3mRNA in skin fibroblasts of scleroderma and normal control were detected by real-time PCR.Results:Through bioinformatics analysis, Smad3mRNA was showed to contain the seed sequence for miR-145binding. Compared with normal control, the expression of miR-145was significantly decreased in skin and skin fibroblast of scleroderma (P<0.05). Smad3mRNA was significantly upregulated in skin tissue and skin fibroblasts of scleroderma (P<0.05). Smad3protein was significantly increased in skin of scleroderma (P<0.05). In the presence of TGF-β, miR-145was significantly increased in skin fibroblasts of scleroderma, and Smad3mRNA was decreased (P<0.05).Conclusion:miR-145may be involved in TGF-β/Smad3profibrotic pathway in scleroderma.