The Role Of MiRNA-21in The Fibrotic Skin Of Systemic Sclerosis And Its Mechanism

Aims:Progressive fibrosis replacement of normal tissue architecture in multiple organs is the main cause of death in systemic sclerosis. It is well documented that transforming growth factor β (TGF-β) plays a fundamental role in the fibrosis process, thus, blockade of TGF-β/Smad signaling pathway which induced persistent fibroblasts activation is the molecular target for anti-fibrotic therapy in systemic sclerosis. Although studies which blockaded of TGF-β/Smad signaling pathway for anti-fibrotic therapy demonstrated good efficacy, TGF-β antagonist didn’t show apparent effects when used for treatment systemic sclerosis patients. The urgent need to find new ways to target TGF-β/Smad signaling pathway. MicroRNAs (miRNAs) bind to the3’untranslated regions (3’UTRs) of target genes and thereby repress the translation of target genes and/or induce the degradation of target gene mRNA. Many miRNAs are regulated by TGF-β, and have their predicted targets genes involved in matrix repairing and remodeling, such as collagens, matrix metalloproteinase, connective tissue growth factor and integrins, and exhibit profibrosis or antifibrosis effects. Our study first investigates the miRNA expression patterns between diffuse and limited cutaneous systemic sclerosis, and search specific miRNAs involved in the pathogenesis of fibrosis (especially in the TGF-β signaling pathway). We investigate the roles of miRNAs in the fibrosis of systemic sclerosis and its mechanism, and identify novel methods for the diagnosis, treatment and prevention of systemic sclerosis.Methods:①Skin biopsies were collected from the lateral forearm of seven systemic sclerosis patients and seven normal controls. Skin biopsies were used for isolating total RNA and protein, culturing primary fibroblasts and preparing for morphology studies. Total RNA was used for miRNA microarray analysis; altered miRNAs were analyzed by computational prediction algorithms, these miRNAs which target genes were involved in the fibrotic pathways were selected. Real-time PCR and in situ hybridization were used to confirm the fibrosis related genes. Real-time PCR and immunohistochemistry were performed to study the expression of target genes both in skin tissues and fibroblasts.②Fibroblasts were stimulated with TGF-β (different concentrations or different time periodsor), real-time PCR or western blot was used to study the expression pattern of miRNAs and taget genes; To indentify miRNAs’directed target, we futher used miRNA-specific mimics or inhibitor modulating miRNA expression and constructed luciferase reporter gene system.③To mimick the systemic sclerosis model, skin fibrosis was induced by local intracutaneous injections bleomycin in mice upper back skin, skin fibrosis was inhibited by peritoneal injection proteasome inhibitor bortezomib; skin tissues were stained by hematoxylin-eosin stain to observe the fibrosis extent; real-time PCR or western blot was used to study the expression of miRNAs, target genes and fibrosis related genes and study the role and mechanism of miRNAs in vivo.Results:(1) miRNA microarray and bioinformatics analysis were used for identifying fibrosis related genes.①Compaired with normal controls,42miRNAs were significantly regulated in the diffuse cutaneous systemic sclerosis skin biopsies,25miRNAs were up-regulated and17were down-regulated,60miRNAs were changed in the limited cutaneous systemic sclerosis skin biopsies,42miRNAs were up-regulated and18miRNAs were up-regulated. A total of21miRNAs were altered similarly both in diffuse and limited cutaneous systemic sclerosis and limited,13miRNAs were up-regulated and8miRNA were down-regulated.②Real-time PCR confirmed that miR-21was increased whereas miR-145and miR-29b were decreased both in the skin tissues and fibroblasts of systemic sclerosis.③The computational predictions indicated SMAD7would be regulated by miR-21, SMAD3would be regulated by miR-145, and COL1A1would be regulated by miR-29b.④Real-time PCR and immunohistochemistry suggested that the expression of Smad7was decreased; The expression of Smad3was increased both in the skin tissues and fibroblasts of systemic sclerosis.(2) The role of miR-21in fibrosis and the relationship between miR-21and Smad7were studied in primary skin fibroblasts in vitro.①After stimulating by TGF-β with different concentrations or different time periods, we found that TGF-β induced the expression of miR-21and fibrosis-related genes, and decreased Smad7expression.②Over-expression of miR-21in fibroblasts decreased the levels of Smad7, whereas knockdown of miR-21increased its expression. Over-expression miR-21exerted profibrosis effect whereas knockdown miR-21exerted antifibrosis effect. Further study using a reporter gene assay demonstrated the Smad7was a direct target of miR-21.(3) The role of miR-21in fibrosis was studied in bleomycin-induced mice vivo.①Histopathology of skin lesions in bleomycin-induced mice was characterized by an excessive deposition of collagens and deep fibrosis, skin fibrosis was ameliorated in bortezomib-treated mice.②In bleomycin-induced group, the expression of miR-21and fibrosis related genes were increased and the expression of Smad7was increased; In bortezomib-treated group, the expression levels of miR-21and fibrosis related genes were decreased and the expression of Smad7was also decreased. Our study only use bortezomib to inhibit fibrosis, and didn’t use miR-21inhibitor which direct inhibits the expression of miR-21in mice, So the relationship between miR-21and Smad7was complicated and affected by many factors.Conclusions:①The miRNA expression pattern in SSc skin tissue is unique.②miR-21, miR-145and miR-29b might play an important role in the pathogenesis of systemic sclerosis.③miR-21exerts profibrotic effect in systemic sclerosis by inhibiting Smad7expression. ④The expression of miR-21, fibrosis related genes and Smad7were consistent, the relationship between miR-21and Smad7was complicated and affected by many factors in vivo.