Activation Of TGF?1/Smad Signal Pathway Mediated By MiR-21 For Denervation-induced Skeletal Muscle Atrophy And Fibrosis

Background Denervation leads to atrophy and fibrosis of intrinsic laryngeal muscle which adversely affect the recovery of laryngeal function following surgical reinnervation for vocal fold paralysis patients in clinic.Abnormal activation of TGF?1/Smad signaling pathway is the key to the atrophy and fibrosis of denervated skeletal muscle.As an important regulatory mechanism in the body's physiological and pathological processes,miRNAs have been increasingly concerned in recent years.Research group of the previous study confirmed that expression of mi R-21 upregulated in skeletal muscle of denervation.Its important role in the fibrosis process of liver,kidney,cardiac muscle and other tissues and organs has been reported in the literature.Objectives To study the effect and mechanism of miR-21 by regulation of TGF?1/Smad signaling pathway on denervation-induced skeletal muscle atrophy and fibrosis.To provide possible therapeutic target for the treatment of skeletal muscle atrophy and fibrosis induced by denervation.Methods 1.We found 20 possible mi RNAs after bioinformatics analysis of key genes in TGF?1/Smad signaling pathway.And we detected the expression level of them in the denervation skeletal muscle.2.We used bioinformatics analysis software,including Targerscan,PicTar and miRanda,to analyze miR-21,then found out the possible target genes on the TGF?1/Smad signaling pathway.The report gene vector pmiRGLO were combined by the candidate target genes,then co transfected mouse fibroblast cells(NIH/3T3)with artificial synthesis mi R-21.The relative fluorescence intensity was detected,QPCR and WB experiments were used to further verify the relative expression.3.We induced activation of mouse fibroblast cells with recombinant TGF?1.The miR-21 expression was successfully reduced by miR-21 inhibition vector(Anti-miR-21).The interference effect of miR-21 and the expression of ?-SMA,p-Smad2/3 and Smad7 were detected by QPCR,immunofluorescence and other techniques.We also performed flow cytometry after transfection with miR-21 mimics and Anti-miR-21 in NIH/3T3 cell.Fluorescence intensity of Smad7 and p-Smad2/3 and nuclear translocation ability of p-Smad2/3 were detected.4.We constructed animal model of sciatic nerve injury.And the miR-21 inhibition vector(pLKO-Anti-miR-21)was constructed.After transfected the vector into mouse gastrocnemius muscle,we killed animals 2 weeks after operation and collected gastrocnemius muscle samples.We detected the expression of mi R-21,Smad7,TGF?1,CTGF,?-SMA and collagen-I by immunofluorescence,Q-PCR and other techniques.Results 1.We found a significant increase in the number of mi RNAs,including mi R-21.2.After bioinformatics analysis,we found out the possible target genes on the TGF?1/Smad signaling pathway were TGF?1,TGF?R2 and Smad7.The relative fluorescence intensity of Smad7 and TGF?R2 decreased significantly.QPCR and WB experiments confirmed that Smad7 and TGF?R2 were direct target gene of miR-21.3.The results showed that the inhibition of mi R-21 could significantly increase the expression of Smad7 and down regulate the expression of p-Smad2/3.Downstream key fibrosis indicators such as ?-SMA expression decreased.The results of flow cytometry showed that the fluorescence intensity of Smad7 decreased after transfection with miR-21,while the fluorescence intensity and nuclear translocation ability of p-Smad2/3 increased.After transfection with Anti-mi R-21,the fluorescence intensity of Smad7 increased and the fluorescence intensity of p-Smad2/3 decreased.It proved that mi R-21 could directly inhibit Smad7 in activated fibroblasts,and promoted the formation and transformation of the p-Smad2/3,then promoted the synthesis and secretion of the fibrosis factors ?-SMA.4.The results showed that after the expression of miR-21 was inhibited,the expression of Smad7 was upregulated and the expression of TGF?1,CTGF,?-SMA and collagen-I were downregulated in the tissue of the mouse muscle damage model.Masson staining showed the degree of atrophy and fibrosis of the gastrocnemius muscle in the miR-21 inhibition group was less than that in the control group It revealed that inhibition of miR-21 could reduced the degree of skeletal muscle atrophy and fibrosis induced by denervation.Conclusion The role of miR-21 in the regulation of skeletal muscle fibrosis induced by denervation was elucidated in this study.The mechanism is miR-21 by inhibiting the expression level of Smad7,inducing the formation and transformation of p-Smad2/3 complex,and promoting the synthesis and secretion of induced fibrosis factors.The results provide a potential therapeutic strategy for the treatment of skeletal muscle atrophy and fibrosis,maintaining the integrity of skeletal muscle structure and function,and promoting the functional recovery of skeletal muscle after nerve repair.