m<sup>6</sup>A recognition protein YTHDF1 ameliorates Duchenne muscular dystrophy and its mechanism

Zheng Yan-Yan; Wang Yan; Li Bei

doi:10.11855/j.issn.0577-7402.1795.2023.0614

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m⁶A recognition protein YTHDF1 ameliorates Duchenne muscular dystrophy and its mechanism

Basic Research | 更新时间：2024-01-04

- m⁶A recognition protein YTHDF1 ameliorates Duchenne muscular dystrophy and its mechanism
- Medical Journal of Chinese People′s Liberation Army Vol. 48, Issue 12, Pages: 1403-1411(2023)
- 作者机构：
  
  西安市儿童医院神经内科，陕西西安 710002
- 作者简介：
  
  E-mail: wxvjd9pq@163.com
- 基金信息：
  
  the Shaanxi Science and Technology Research Project for Social Development(2015SF214);the Scientific Research Project of Xi'an Children's Hospital(2022G03)
- DOI：10.11855/j.issn.0577-7402.1795.2023.0614
  CLC：
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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.

Zheng Yan-Yan,Wang Yan,Li Bei.m⁶A recognition protein YTHDF1 ameliorates Duchenne muscular dystrophy and its mechanism[J].Medical Journal of Chinese People′s Liberation Army,2023,48(12):1403-1411.
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. DOI： 10.11855/j.issn.0577-7402.1795.2023.0614.

Zheng Yan-Yan,Wang Yan,Li Bei.m⁶A recognition protein YTHDF1 ameliorates Duchenne muscular dystrophy and its mechanism[J].Medical Journal of Chinese People′s Liberation Army,2023,48(12):1403-1411. DOI： 10.11855/j.issn.0577-7402.1795.2023.0614.

摘要

目的,2,探讨m,6,A识别蛋白YTHDF1对杜氏肌营养不良症(DMD)的改善作用及其潜在机制。,方法,2,(1)收集2016年9月－2017年6月于西安市儿童医院确诊并进行手术的38例DMD患者(设为DMD组)和33例接受骨科手术且与DMD患者年龄匹配(2~3岁)的对照患者(设为对照组)的肌肉组织和血液样本，采用RT-qPCR和Western blotting检测肌肉组织中YTHDF1和抗肌萎缩蛋白(dystrophin)的表达水平；ELISA检测血清肌酸磷酸激酶(CPK)含量。(2)取骨骼肌成肌细胞SkMCs，设置空载体组(转染Scrambled阴性对照)、AAV-YTHDF1组(转染AAV-YTHDF1表达载体)与Scrambled组(转染NC siRNA)、si-YTHDF1组(转染,YTHDF1, siRNA)，采用EdU检测细胞增殖能力；Western blotting检测dystrophin、肌细胞生成素(MyoG)和肌球蛋白重链(MHC)蛋白的表达水平；RIP验证YTHDF1与Yes关联蛋白1(YAP1) mRNA的相互作用，及,YAP1 ,mRNA上m,6,A位点的修饰水平。(3)30只Mdx小鼠随机分为空载体组(,n,=15，腹腔注射腺病毒空载体)与AAV-YTHDF1组(,n,=15，腹腔注射含YTHDF1过表达载体的腺病毒)，测量小鼠体重、肌肉和器官湿重、纤维直径及纤维类型。采用Western blotting检测YTHDF1、dystrophin蛋白的表达水平，HE染色观察腓肠肌和股四头肌病理学变化情况。,结果,2,(1)与对照组比较，DMD组患者肌肉组织中YTHDF1、dystrophin蛋白表达水平明显降低(,P,<,0.01)，血清CPK含量明显增加(,P,<,0.0001)。(2)与空载体组比较，AAV-YTHDF1组EdU阳性细胞率以及dystrophin、MyoG、MHC蛋白表达水平明显升高(,P,<,0.01)。过表达YTHDF1延长了,YAP1, mRNA的半衰期，提高了,YAP1, mRNA的稳定性。与Scrambled组比较，si-YTHDF1组EdU阳性细胞率以及dystrophin、MyoG、MHC蛋白表达水平明显降低(,P,<,0.05或,P,<,0.01)。(3)与空载体组比较，AAV-YTHDF1组小鼠发育迅速，腓肠肌、股四头肌、三头肌肌肉重量增加(,P<,0.05)，腹股沟、性腺或腹膜后脂肪垫重量无明显差异(,P>,0.05)，腓肠肌和股四头肌的纤维面积明显增大(,P,<,0.05)。,结论,2,YTHDF1可通过识别,YAP1, mRNA的m,6,A修饰调节,YAP1, mRNA的稳定性，进而促进YAP1/dystrophin介导的肌细胞增殖，抑制DMD进展。

Abstract

Objective,2,To investigate the improvement effect and potential mechanism of m,6,A recognition protein YTHDF1 on Duchenne muscular dystrophy (DMD).,Methods,2,(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 m,6,A 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,2,(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,2,YTHDF1 regulates the stability of ,YAP1, mRNA by recognizing the m,6,A 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YTHDF1m6A增殖

Keywords

Duchenne muscular dystrophyYTHDF1m6Aproliferation

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⁰

m6A recognition protein YTHDF1 ameliorates Duchenne muscular dystrophy and its mechanism

DOI：10.11855/j.issn.0577-7402.1795.2023.0614

摘要

Abstract

关键词

Keywords

references

m⁶A recognition protein YTHDF1 ameliorates Duchenne muscular dystrophy and its mechanism