调控TGFβ1/SMAD通路的microRNAs在失神经支配骨骼肌中的表达

Expression of microRNAs potentially targeting TGFβ1/SMAD signaling pathway in denervated skeletal muscle

目的：本研究拟探讨骨骼肌失神经支配后差异性表达的microRNAs,并明确其是否参与调控TGFβ1/SMAD信号通路.方法：C57/BL6J小鼠,随机分为正常对照组和实验组,实验组小鼠切断右下肢坐骨神经为手术组,左下肢游离坐骨神经作为假手术组.4周后处死小鼠取腓肠肌Masson染色观察肌纤维形态变化,比较各组肌纤维横截面积.以TGFβ1/SMAD为靶基因,通过生物信息学分析寻找到18个可能的miRNAs指标.定量PCR检测其表达,并针对表达升高的microRNAs采用荧光素酶报告基因实验确认其靶基因.结果：手术组肌纤维横截面积比对照组明显缩小,对照组与假手术组肌肉之间的差异无统计学意义.在失神经骨骼肌中特异性高表达的有miR-424、miR-744、miR-15a.在C2C12细胞系中行报告基因实验显示,与对照组比较,转染miR-744后,SMAD3的荧光素酶强度下降;转染miR-15a后,TGFβ1的荧光素酶强度下降;转染miR-424后,SMAD3的荧光素酶强度变化无统计学意义.PCR验证microRNAs的靶基因显示,转染miR-744、miR-15a的模拟物后,分别对应的SMAD3、TGFβ1表达下调,差异有统计学意义.结论：miR-424、miR-744、miR-15a可能参与调控失神经支配导致的骨骼肌萎缩纤维化,其中miR-744的靶基因是SMAD3,miR-15a的靶基因是TGFβ1.

Objective To explore the expression of microRNAs involved in the regulation of TGF1/SMAD signaling pathway indenervated gastrocnemius muscle in mice. Methods： C57/B L6J mice were randomly divided into experimental group and controlgroup. For experimental group the right sciatic nerve was cut off and the lett sciatic nerve was received sham operation. After 4weeks, the mice were sacrificed and the gastrocnemius muscle was removed We did muscle MASSON staining, then observedthe morphological changes of muscle fibers and compared their cross-sectional area between groups. Genes in TGFβ1/SMAD signaling pathwyy were targeted and 18 possible miRNAs were found, based on bioinformatics analysis. Expression of these mRNAs was detected by Q-PCR. Several miRNAs expression increased significantly. Luciferase reporter gene assay in C2C12 cell mode confirmed the miRNAs and targeted genes related Results ： The cross-sectional area of the muscle fibers in the denervation group was significantly smaller than that in the control group. There was no signifgroup and the sham operation group. 3 miRNAs expressed high levels specificially in the denervated muscle： miR -4 2 4 , miR-744, miR-15a Luciferase reporter gene assay in C2C12 cell mode showed, compared with the control activity of SMAD3 was decreased after transfected miR-7 4 4. The luciferase activity of TGFβ1 was decreased after transfected miR-15a. The luciferase activity of SMAD3 was no significant different ater transfected miR-424. PCR verified target genes of 2 miRNAs. After transfection of miR-744, miR-15a, the corresponding expression of SMAD3 and Conclusion ： MiR-424, miR-7 44 and miR-15a may be involved in the regulation of denervation-induced muscle atrophy andfibrosis. The target gene of miR-744 was SMAD3. The target gene of miR-15a was TGF＂1.