Expression And Regulation Of MiRNA-21 In Human Hypertrophic Scar Fibroblasts

Background:Hypertrophic scar is a common complication of severe trauma, burns and surgical operation, resulting in dysfunction and deformity of corresponding parts for patients, which decreased quality of life in these patients. In the formation and development of hypertrophic scar, skin tissue is mainly repaired by a large number of fibroblast proliferations and inhibition of apoptosis. The equilibrium between positive and negative cytokines stimulated by inflammatory, and between synthesis and metabolism of extracellular matrix, was broken, which was the biological procedure of hypertrophic scarring. Although there are many studies confirmed that there are many differences between the hypertrophic scar fibroblasts and the normal skin fibroblasts, but the pathogenesis is not completely elucidated.MicroRNA(miRNA) as an endogenous RNA, widely participates in cell proliferation, differentiation、apoptosis and many other biological processes. Studies have shown that miRNA plays an important role in abnormal cell proliferation, such as the tumor happen and fibrosis development. Our previous studies have shown that miRNAs have differential expression in human hypertrophic scar, especially miRNA-21 had significant change, but the specific biological effects and mechanism is not very clear. Therefore, we test the expression and change of miRNA21, TGF-β1, PDCD4, Smad7, collagen type Ⅰ and collagen type Ⅲ in hypertrophic scar fibroblasts with real-time fluorescence quantitative polymerase chain reaction, Western blotting, immunocytochemistry. Those studies are attempted to deeply elucidate the molecular mechanisms of hypertrophic scar, providing a theoretical basis and experimental basis for further clinical application of miRNA21. Objection:To observe the expression and change of miRNA21, TGF-β1, PDCD4, Smad7, collagen typeⅠand collagen type Ⅲ in hypertrophic scar fibroblasts in vitro, and to look for the molecular mechanisms and novel therapeutic molecule target of hypertrophic scar. Methods and materials:(1) Hypertrophic scar fibroblasts and the normal skin fibroblasts were cultured in vitro, respectively, with the method which combined tissue explants adherent and enzyme digestion. Then we test the expression and change of miRNA21, TGF-β1, PDCD4, Smad7, Ⅰ type collagen and Ⅲ type collagen between hypertrophic scar fibroblasts and the normal skin fibroblasts with real-time fluorescence quantitative polymerase chain reaction, Western blotting, immunocytochemistry.(2) mi RNA21 mimics were transfected into human hypertrophic scar fibroblasts by liposome transfection technology in vitro. Then, using real-time fluorescence quantitative polymerase chain reaction, Western blotting, we observe the expression and change of miRNA21, TGF-β1, PDCD4, Smad7, Ⅰ type collagen and Ⅲ type collagen.(3)miRNA21 inhibitors were transfected into human hypertrophic scar fibroblasts by liposome transfection technology in vitro,then we test the expression and change of mi RNA21, TGF-β1, PDCD4, Smad7, collagen typeⅠand collagen type Ⅲ between hypertrophic scar fibroblasts and the normal skin fibroblasts with real-time fluorescence quantitative polymerase chain reaction, Western blotting. Results:(1) Campared with the normal skin fibroblasts, the expression of mi RNA21, TGF-β1, collagen typeⅠand collagen type Ⅲwere increased while expression of PDCD was decreased in human hypertrophic scar fibroblasts in vitro. The expression level of miRNA21 was in significant negative correlation with the expression level of PDCD4 and in positive correlation with the expression level of TGF-β1.(2)Campared with the control group,the expression level of mRNA and protein of mi RNA21, TGF-β1, collagen typeⅠand collagen type Ⅲ were increased while expression level of mRNA and protein of PDCD4 and Smad7 were decreased in human hypertrophic scar fibroblasts transfection with mi RNA21 mimics.(3) Campared with the control group,the expression level of mRNA and protein of PDCD4 and Smad7 were increased while expression level of mRNA and protein of mi RNA21, TGF-β1, collagen typeⅠand collagen type Ⅲ were decreased in human hypertrophic scar fibroblasts transfection with miRNA21 inhitors.(1)The expression level of miRNA21, TGF-β1, collagen typeⅠ and collagen type Ⅲ in cultured hypertrophic scar fibroblasts was significantly higher than that in normal skin fibroblasts, and the expression level of PDCD4 and Smad7 in human hypertrophic scar fibroblasts was significantly lower than that in normal skin fibroblasts, which may involve in the process of hypertrophic scar formation and evolution.(2) The up-regulated expression of miRNA21 can promote the expression of mi RNA21, TGF-β1, collagen typeⅠ and collagen type Ⅲ, inhibit the expression of PDCD4 and Smad7 in human hypertrophic scar fibroblasts, which may be one of the important mechanisms of hypertrophic scar formation.(3) The down-regulated expression of miRNA21 can promote the expression of PDCD4 and Smad7, inhibit the expression of mi RNA21, TGF-β1, collagen typeⅠand collagen type Ⅲ in human hypertrophic scar fibroblasts, indicating that miRNA21 may be a potential therapeutic molecule target of hypertrophic scar.