解放军医学杂志2023，Vol.48Issue(12)：1403-1411,9.DOI:10.11855/j.issn.0577-7402.1795.2023.0614
m6A识别蛋白YTHDF1对杜氏肌营养不良症的改善作用及其机制
m6A recognition protein YTHDF1 ameliorates Duchenne muscular dystrophy and its mechanism
摘要
Abstract
Objective To investigate the improvement effect and potential mechanism of m6A recognition protein YTHDF1 on Duchenne muscular dystrophy(DMD).Methods(1)We collected muscle tissue and peripheral blood from DMD patients(38 confirmed patients with surgery at Xi'an Children's Hospital from September 2016 to June 2017,DMD group)and non-DMD patients with orthopedic surgery(33 age-matched patients,2-3 years old,control group).The expression levels of YTHDF1 and dystrophin in the muscle tissues were quantified by RT-qPCR and Western blotting.The serum creatine phosphokinase(CPK)content was determined using ELISA.(2)Take SkMC from skeletal muscle myoblasts and set up empty vector group(transfected with scrambled negative control),AAV-YTHDF1 group transfected with YTHDF1 overexpression vector and scrambled group(transfected with NC siRNA)and si-YTHDF1 group(transfected with YTHDF1 siRNA).Cell proliferation was analyzed using an EdU assay.The expressions of dystrophin,myogenin(MyoG),and myosin heavy chain(MHC)proteins were measured by Western blotting.A RIP assay was used to verify the interaction between YTHDF1 and YES-associated protein 1(YAP1)mRNA and investigate the modification level of the m6A site on YAP1 mRNA.(3)Thirty Mdx mice were randomly divided into:the empty vector group(n=15,intraperitoneal injection of empty adenoviral empty vector)and the AAV-YTHDF1 group(n=15,intraperitoneal injection of YTHDF1 overexpression adenovirus vector).We profiled the body weights,muscle and organ wet weights,fiber diameter,and fiber type of the mice.The protein levels of YTHDF1 and dystrophin were detected using Western blotting.We used HE staining to observe the pathological changes in the gastrocnemius and quadriceps femoris muscles.Results(1)Compared with control group,the expression of YTHDF1 and dystrophin was significantly lower in DMD patients(P<0.01),and the serum CPK content was significantly increased(P<0.0001).(2)Compared with empty vector group,the EdU positive rate and the expression of dystrophin,MyoG,and MHC proteins significantly increased(P<0.01).In addition,overexpression of YTHDF1 prolonged the half-life and improved the stability of YAP1 mRNA.Compared with Scramble group,the EdU-positive rate and the expression of dystrophin,MyoG,and MHC proteins significantly decreased(P<0.05 or P<0.01).(3)Compared with empty vector group,the mice in AAV-YTHDF1 group developed more rapidly with higher muscle mass in the gastrocnemius muscle,quadriceps muscle,and triceps(P<0.05).We observed no difference in the weights of inguinal,gonadal,or retroperitoneal fat pads and a significant increase in the fiber areas of gastrocnemius and quadriceps femoris(P>0.05).Conclusion YTHDF1 regulates the stability of YAP1 mRNA by recognizing the m6A modification of YAP1 mRNA and thus promotes YAP1/dystrophin-mediated myocyte proliferation to inhibit DMD progression.关键词
杜氏肌营养不良症/YTHDF1/m6A/增殖Key words
Duchenne muscular dystrophy/YTHDF1/m6A/proliferation分类
医药卫生引用本文复制引用
郑妍妍,王燕,李蓓..m6A识别蛋白YTHDF1对杜氏肌营养不良症的改善作用及其机制[J].解放军医学杂志,2023,48(12):1403-1411,9.基金项目
This work was supported by the Shaanxi Science and Technology Research Project for Social Development(2015SF214),and the Scientific Research Project of Xi'an Children's Hospital(2022G03) 陕西省社会发展科技攻关项目(2015SF214) （2015SF214）
西安市儿童医院院级科研项目(2022G03) （2022G03）
