外源性miR206抑制Myostatin表达影响大鼠失神经性肌萎缩的进程

Effect of exogenous miR206 on the development of denervated muscular atrophy by inhibiting the expression of myostatin of rats

目的探讨miR206在骨骼肌失神经萎缩中的作用机制。方法 2020年9月至2020年12月,共选取40只SPF级SD大鼠进行本研究。应用16只SD大鼠剪除后肢坐骨神经1 cm建立腓肠肌失神经支配模型,分别在腓肠肌失神经支配时间0、3、7、14 d分为4组取材,每组4只,将样本进行湿重比测定观察无干预下大鼠肌肉失神经萎缩的变化。另24只大鼠同样建立腓肠肌失神经支配模型,并分为3组,每组8只,分别为:去神经加miR206转染组、去神经加对照转染组和假手术组,于失神经支配7 d后取材。将样本进行湿重比测定和Masson染色下肌纤维横截面积测量来评估肌肉萎缩的程度,并通过Western blot和qPCR检测腓肠肌中Myostatin和miR206的表达水平来评估二者的关系。细胞实验中,以C2C12细胞为对象,将带Myostatin(MSTN)mRNA 3’UTR端的荧光质粒和带mut MSTN mRNA 3’UTR端的荧光质粒,分别与mmu-miR206、mmu-miR206抑制剂、NC和NC抑制剂共转染,通过荧光素酶报告分析,对比各组的荧光强度来验证miR206和Myostatin的靶向关系。两组数据间比较采用t检验,多组数据结果间比较采用单因素方差分析,若组间差异有统计学意义,进一步以Bonferroni法进行两两比较,P<0.05为差异有统计学意义。结果在去神经组观察中发现,大鼠腓肠肌湿重比整体随失神经支配时间延长而降低,于第7天时最为明显。去神经加miR206转染组、去神经加对照转染组和假手术组的腓肠肌湿重比分别为0.63±0.04、0.51±0.02和1.05±0.02,肌纤维横截面积分别为(761.30±21.79)、(640.30±30.31)和(1 066.00±51.65)μm^(2),各组间差异均有统计学意义(P<0.05)。Western blot结果显示,去神经加miR206转染组中Myostatin的蛋白相对表达量低于去神经加对照转染组,qPCR结果显示两组Myostatin的mRNA相对表达量分别为0.57±0.04和0.81±0.04。无论蛋白和mRNA,去神经加miR206转染组中的相对表达量均低于去神经加对照转染组,差异均有统计学意义(P<0.05)。荧光素酶报告分析显示, mmu-miR206共转染MSTN荧光质粒组的荧光相对强度是(0.26±0.07),显著低于其余各组,差异均有统计学意义(P<0.05)。结论外源性miR206能通过特定的作用靶点来抑制Myostatin的表达,进而在大鼠腓肠肌失神经性肌萎缩中发挥积极的作用,并且二者间存在靶向作用的可能。

Objective To investigate the effects and mechanism of miR206 in rat model of denervated muscular atrophy.Methods From September,2020 to December,2020,a total of 40 rats were selected for this study.Denervated muscular atrophy model was established on 16 SPF Sprague-Dawley rats,by removing 1 cm in length of sciatic nerve.The rats were classified into 4 groups according to the sampling time:0 d,3 d,7 d and 14 d(4 rats per group).The other 24 rats were also established into denervated skeletal muscle atrophy models and assigned into 3 groups:denervation add miR206 group,denervation add NC transfection reagent group,and sham-operated group(n=8 in each group).After sampling,the area of cross section of the gastrocnemius muscle and gastrocnemius muscle mass were measured to evaluate muscle atrophy.The mRNA and protein expression of myostatin were determined by real-time PCR and Western blot.Combining with luciferase report to explore the underlying mechanism of miR206,the t—test and oneway ANOVA were used for data analysis used in this study.In one-way ANOVA analysis,if the difference between groups was statistically significant,Bonferroni method would be used for further comparing of all pairs.P<0.05 was considered statistically significant.Results After excision of a part of sciatic nerve of rat models,gastrocnemius muscle mass of denervation plus miR206 group,denervation plus NC transfection reagent group and sham-operated group were:(0.63±0.04),(0.51±0.02)and(1.05±0.02),respectively.The cross section areas of gastrocnemius muscle in each groups were:(761.30±21.79)μm^(2),(640.30±30.31)μm^(2)and(1066.00±51.65)μm^(2),respectively(P<0.05).Myostatin mRNA expression showed lower in miR206 group than in NC group tested by Western blot,which were(0.57±0.04)in miR206 and(0.81±0.04)in NC group tested by qPCR(P<0.05).The protein expression measured by Western blot test revealed same expression pattern as mRNA expression pattern.The different of relative expression between miR206 group and NC group(P<0.05).Finally,in the mmu-miR206 co-transfected with the MSTN 3TJTR-luciferase sensor group,the relative luciferase activity was measured at 0.26±0.07 and it was significant lower than any other groups(P<0.05).Conclusion The miR206 can counteract denervated skeletomuscular atrophy through down regulating the myostatin expression.Myostatin is a new discovered target gene of miR206.