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Article(id=1199688709563052594, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1199688705905623579, articleNumber=null, orderNo=null, doi=10.11855/j.issn.0577-7402.1795.2023.0614, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1661443200000, receivedDateStr=2022-08-26, revisedDate=null, revisedDateStr=null, acceptedDate=1667577600000, acceptedDateStr=2022-11-05, onlineDate=1763957677497, onlineDateStr=2025-11-24, pubDate=1703692800000, pubDateStr=2023-12-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1763957677497, onlineIssueDateStr=2025-11-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1763957677497, creator=13701087609, updateTime=1763957677497, updator=13701087609, issue=Issue{id=1199688705905623579, tenantId=1146029695717560320, journalId=1189873630562394117, year='2023', volume='48', issue='12', pageStart='1359', pageEnd='1491', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1763957676626, creator=13701087609, updateTime=1763958367038, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1199691601774739748, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1199688705905623579, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1199691601774739749, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1199688705905623579, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1403, endPage=1411, ext={EN=ArticleExt(id=1199688710007648830, articleId=1199688709563052594, tenantId=1146029695717560320, journalId=1189873630562394117, language=EN, title=m6A recognition protein YTHDF1 ameliorates Duchenne muscular dystrophy and its mechanism, columnId=1190310110212751762, journalTitle=Medical Journal of Chinese People’s Liberation Army, columnName=Basic Research, runingTitle=null, highlight=null, articleAbstract=

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.

, correspAuthors=Bei Li, authorNote=null, correspAuthorsNote=
E-mail:
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目的 探讨m6A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 (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m6A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 (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 YTHDF1可通过识别YAP1 mRNA的m6A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。

, correspAuthors=李蓓, authorNote=null, correspAuthorsNote=
李蓓，E-mail：
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郑妍妍，硕士研究生，主要从事小儿神经系统疾病的诊断与治疗研究

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郑妍妍，硕士研究生，主要从事小儿神经系统疾病的诊断与治疗研究

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郑妍妍，硕士研究生，主要从事小儿神经系统疾病的诊断与治疗研究

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dystrophin. 抗肌萎缩蛋白；A. 两组肌肉组织中YTHDF1 mRNA表达水平；B. 两组肌肉组织中YTHDF1蛋白表达水平(n=5)；C. 两组血清肌酸磷酸激酶(CPK)含量；D. 杜氏肌营养不良症(DMD)患者YTHDF1蛋白表达水平与血清CPK含量的相关性；E. 两组肌肉组织中dystrophin蛋白表达水平；**P<0.01；***P<0.001

, figureFileSmall=n57cCOxDavMRr2EsXw28bw==, figureFileBig=rWBr/bMzfYMn8JrSUy4XGQ==, tableContent=null), ArticleFig(id=1199688714566857542, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1199688709563052594, language=EN, label=Fig.2, caption=Effects of overexpression of YTHDF1 on myocyte proliferation and expression levels of dystrophin, MyoG and MHC, figureFileSmall=Y5GKQwT0rBJr67cVNLZVWg==, figureFileBig=grC3XZDmK/0kEdwxNAd0mQ==, tableContent=null), ArticleFig(id=1199688714705269577, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1199688709563052594, language=CN, label=图2, caption=过表达YTHDF1对肌细胞增殖及dystrophin、MyoG和MHC蛋白表达的影响

dystrophin. 抗肌萎缩蛋白；MyoG. 肌细胞生成素；MHC. 肌球蛋白重链；A. AAV-YTHDF1转染SkMC细胞后，检测YTHDF1过表达效率(n=5)；B. 过表达YTHDF1后SkMC细胞中dystrophin、MyoG和MHC蛋白表达水平(n=5)；C. EdU检测过表达YTHDF1后SkMC细胞增殖情况(n=5)；**P<0.01

, figureFileSmall=Y5GKQwT0rBJr67cVNLZVWg==, figureFileBig=grC3XZDmK/0kEdwxNAd0mQ==, tableContent=null), ArticleFig(id=1199688714814321487, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1199688709563052594, language=EN, label=Fig.3, caption=Effects of YTHDF1 knockdown on myocyte proliferation and expression levels of dystrophin, MyoG and MHC, figureFileSmall=vg78A5F4VXq1ScR2u8GVHg==, figureFileBig=5i9EQ7G3EKm7xPnLMEs0oA==, tableContent=null), ArticleFig(id=1199688714919179092, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1199688709563052594, language=CN, label=图3, caption=敲低YTHDF1对肌细胞增殖和dystrophin、MyoG和MHC蛋白表达的影响

dystrophin. 抗肌萎缩蛋白；MyoG. 肌细胞生成素；MHC. 肌球蛋白重链；A. si-YTHDF1转染SkMC细胞后，Western blotting检测YTHDF1敲低效率(n=5)；B. 敲低YTHDF1后SkMC细胞中dystrophin、MyoG和MHC蛋白表达水平(n=5)；C. EdU检测敲低YTHDF1后SkMC细胞增殖情况(n=5)；*P<0.05，**P<0.01

, figureFileSmall=vg78A5F4VXq1ScR2u8GVHg==, figureFileBig=5i9EQ7G3EKm7xPnLMEs0oA==, tableContent=null), ArticleFig(id=1199688715128894297, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1199688709563052594, language=EN, label=Fig.4, caption=The interaction between YTHDF1 protein and YAP1 mRNA, figureFileSmall=TgfJ1N/LQNMy+4/5IKbJdA==, figureFileBig=JcPT7ZNr2tY5Mvv+VHpKMw==, tableContent=null), ArticleFig(id=1199688715246334814, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1199688709563052594, language=CN, label=图4, caption=YTHDF1蛋白与YAP1 mRNA的相互作用

A. SRAMP在线数据库预测显示YAP1 mRNA上存在m6A修饰位点；B. RIP验证YTHDF1与YAP1 mRNA的相互结合；C. RIP验证YAP1 mRNA上m6A的修饰水平；***P<0.001

, figureFileSmall=TgfJ1N/LQNMy+4/5IKbJdA==, figureFileBig=JcPT7ZNr2tY5Mvv+VHpKMw==, tableContent=null), ArticleFig(id=1199688715388941159, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1199688709563052594, language=EN, label=Fig.5, caption=Effect of YTHDF1 on YAP1 expression and stability of YAP1 mRNA, figureFileSmall=iX80Xwo/G05diOeTUlpGkQ==, figureFileBig=sYw5Dv99xNzlOBsH/i2zJQ==, tableContent=null), ArticleFig(id=1199688715510575983, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1199688709563052594, language=CN, label=图5, caption=YTHDF1对YAP1表达和YAP1 mRNA稳定性的影响

YAP1. Yes1相关转录调节因子；A. AAV-YTHDF1和si-YTHDF1转染对数生长期SkMC细胞，Western blotting检测YAP1蛋白表达水平(n=5)；B. AAV-YTHDF1和空载体转染放线菌素D处理的SkMC细胞，RT-qPCR检测YAP1 mRNA的稳定性(n=5)；**P<0.01，***P<0.001

, figureFileSmall=iX80Xwo/G05diOeTUlpGkQ==, figureFileBig=sYw5Dv99xNzlOBsH/i2zJQ==, tableContent=null), ArticleFig(id=1199688715619627890, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1199688709563052594, language=EN, label=Fig.6, caption=Effect of YTHDF1 on muscle growth and muscle mass in DMD mice, figureFileSmall=1sBDGdxDpvA5hYf1VZaakg==, figureFileBig=3B/Fk+9gAjzx/dE0BuvC8w==, tableContent=null), ArticleFig(id=1199688715732874103, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1199688709563052594, language=CN, label=图6, caption=YTHDF1对DMD小鼠肌肉生长和肌肉质量的影响

DMD. 杜氏肌营养不良症；dystrophin. 抗肌萎缩蛋白；A. AAV-YTHDF1组和空载体组小鼠初次注射至注射20周时体重分布(n=15)，与空载体组比较，**P<0.01；B. Western blotting检测小鼠肌肉组织中YTHDF1和dystrophin蛋白表达水平(n=5)；C. 小鼠腓肠肌、四头肌、三头肌肌肉及腹股沟、性腺或腹膜后脂肪垫重量(n=5)；D. 小鼠腓肠肌和股四头肌HE染色(n=5)；*P<0.05，**P<0.01

, figureFileSmall=1sBDGdxDpvA5hYf1VZaakg==, figureFileBig=3B/Fk+9gAjzx/dE0BuvC8w==, tableContent=null), ArticleFig(id=1199688715867091836, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1199688709563052594, language=EN, label=Tab.1, caption=

Primer sequences of RT-qPCR

, figureFileSmall=null, figureFileBig=null, tableContent=
基因上游引物下游引物
YTHDF15'-ATGTCGGCCACCAGCGTGGACA-3'5'-TCATTGTTTGTTTCGACTCTGC-3'
YAP15'-TAGCCCTGCGTAGCCAGTTA-3'5'-TCATGCTTAGTCCACTGTCTGT-3'
GAPDH5'-TCTTAAGAAGACGACGGCTTCAG-3'5'-TTGCTCTCTCACTTGTCCTCGAT-3'
), ArticleFig(id=1199688715967755134, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1199688709563052594, language=CN, label=表1, caption=

RT-qPCR引物序列

, figureFileSmall=null, figureFileBig=null, tableContent=
基因上游引物下游引物
YTHDF15'-ATGTCGGCCACCAGCGTGGACA-3'5'-TCATTGTTTGTTTCGACTCTGC-3'
YAP15'-TAGCCCTGCGTAGCCAGTTA-3'5'-TCATGCTTAGTCCACTGTCTGT-3'
GAPDH5'-TCTTAAGAAGACGACGGCTTCAG-3'5'-TTGCTCTCTCACTTGTCCTCGAT-3'
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m6A识别蛋白YTHDF1对杜氏肌营养不良症的改善作用及其机制
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郑妍妍 , 王燕 , 李蓓 *
解放军医学杂志 | 基础研究 2023,48(12): 1403-1411
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解放军医学杂志 | 基础研究 2023, 48(12): 1403-1411
m6A识别蛋白YTHDF1对杜氏肌营养不良症的改善作用及其机制
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郑妍妍, 王燕, 李蓓*
作者信息
  • 西安市儿童医院神经内科，陕西西安 710002

    • 郑妍妍，硕士研究生，主要从事小儿神经系统疾病的诊断与治疗研究

通讯作者:

李蓓，E-mail：
m6A recognition protein YTHDF1 ameliorates Duchenne muscular dystrophy and its mechanism
Yan-Yan Zheng, Yan Wang, Bei Li*
Affiliations
  • Department of Neurology, Xi'an Children's Hospital, Xi'an, Shaanxi 710002, China
出版时间: 2023-12-28 doi: 10.11855/j.issn.0577-7402.1795.2023.0614
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目的 探讨m6A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 (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m6A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 (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 YTHDF1可通过识别YAP1 mRNA的m6A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。

杜氏肌营养不良症  /  YTHDF1  /  m6A  /  增殖

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.

Duchenne muscular dystrophy  /  YTHDF1  /  m6A  /  proliferation
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. 解放军医学杂志, 2023 , 48 (12) : 1403 -1411 . DOI: 10.11855/j.issn.0577-7402.1795.2023.0614
Yan-Yan Zheng, Yan Wang, Bei Li. 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
正文
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杜氏肌营养不良症(Duchenne muscular dystrophy，DMD)是一种X染色体连锁的隐性遗传病，由编码抗肌萎缩蛋白(dystrophin)的基因突变所致。分裂的成肌细胞是肌肉生长和维持所必需的，而成肌细胞的有限生长能力与DMD的进行性肌肉退化特征直接相关[1-2]。据统计显示，全球新生男婴DMD患病率为1/3500[3]，而目前临床尚无有效疗法。因此，研究DMD发生和发展的机制对实现该病的早期诊断、干预具有重要的临床意义。DMD基因位于染色体Xp21，编码含有3685个氨基酸的dystrophin(427 kD)。肌细胞膜表面存在抗肌萎缩-糖蛋白复合物(dystrophin-associated protein complex，DAPC)，可维持细胞结构的完整性和细胞膜内外的传递运输功能，而dystrophin是DAPC的重要组成部分[4]。N6-甲基腺苷(m6A)修饰是真核生物RNA中最广泛的内部修饰，参与多个生物学过程。不同的阅读蛋白(包括YTHDF、YTHDC和IGFBP等家族蛋白)可特异性识别m6A修饰，并调节RNA的稳定性[5]。研究发现，m6A修饰在肌肉干细胞维持、肌细胞增殖和分化中发挥重要作用[6]。Yes1相关转录调节因子(Yes1 associated transcriptional regulator，YAP1)是一种转录辅助因子，可促进细胞增殖以及调节器官发育和再生，最近研究发现，在DMD心脏组织中YAP1活性明显降低；而抑制YAP1活性可明显降低DMD多能干细胞衍生的心肌细胞增殖，诱导DMD心肌病的发生[7]，但YAP1是否通过调节m6A修饰水平调控肌细胞增殖尚不明确。本研究通过检测DMD患者和健康人群肌肉组织中YTHDF1和dystrophin的表达水平，观察过表达或干扰YTHDF1对人骨骼肌成肌细胞增殖的影响，探究YTHDF1对DMD中成肌细胞生物学行为的影响及其潜在作用机制。
1 材料与方法
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1.1 主要试剂及仪器
胎牛血清(fetal bovine serum，FBS)、Ham's F-10培养基购自美国Gibco公司；Lipofectamine 3000转染试剂、DMEM培养基、青霉素、链霉素、庆大霉素购自美国Invitrogen公司；人表皮生长因子(human epidermal growth factor，hEGF)、地塞米松、L-谷氨酰胺、胰蛋白酶-EDTA溶液购自美国Sigma公司；YTHDF1过表达质粒AAV-YTHDF1及YTHDF1小干扰RNA(si-YTHDF1)购自美国Addgene公司；PrimeScript RT-PCR试剂盒购自日本TaKaRa公司；BCA蛋白含量测定试剂盒、EdU试剂盒购自美国Thermo Scientific公司；磷酸肌酸激酶(creative phospho kinase，CPK) ELISA试剂盒购自上海科顺生物科技有限公司；anti-YTHDF1抗体、anti-dystrophin抗体、anti-YAP1抗体、anti-肌细胞生成素(myogenin，MyoG)抗体、anti-肌球蛋白重链(myosin heavy chain，MHC)抗体、anti-m6A抗体购自英国Abcam公司；Magna RIP RNA结合蛋白免疫沉淀试剂盒、Magna甲基化RNA免疫沉淀(MeRIP) m6A试剂盒购自美国Millipore公司；放线菌素D购自武汉艾美捷科技有限公司。实时荧光定量PCR仪购自德国Eppendorf公司；Thermo Varioskan™ LUX多功能酶标仪、NanoDrop 2000分光光度仪购自美国Thermo Fisher Scientific公司；Western blotting转膜仪购自北京凯元信瑞仪器有限公司；蛋白电泳仪及配套电泳槽购自美国Bio-Rad公司；凝胶成像仪购自英国Uvitec Cambridge公司；倒置普通光学显微镜购自日本Olympus公司。
1.2 标本收集
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)的患者(未患有其他干扰性疾病，设为对照组)的肌肉组织和血液样本(入院时采集，于-80 ℃超低温冰箱保存备用)。纳入标准：(1)经病理和(或)基因检测确诊为DMD；(2)年龄<18岁。排除标准：(1)不能配合完成检查；(2)病理及影像资料不完整；(3)存在MRI检查禁忌证。本研究经西安市儿童医院伦理委员会批准(20160918-1F)，所有受试者或监护人均签署知情同意书。
1.2.1 RT-qPCR检测YTHDF1 mRNA水平
使用Trizol试剂提取肌肉组织总RNA，并用Prime Script RT试剂盒反转录成cDNA。使用SYBR Premix Ex Taq Ⅱ在ABI 7500实时PCR系统中进行实时定量反转录PCR。PCR反应体系(20 μl)：cDNA 2 μl、Taq聚合酶0.4 μl、上下游引物各0.8 μl、ddH2O 6 μl、2×SYBR绿色PCR混合液10 μl。PCR反应条件：95 ℃ 1 min；95 ℃ 20 s、56 ℃ 10 s、72 ℃ 15 s，35个循环。以GAPDH为内参照，采用2-ΔΔCt法计算YTHDF1 mRNA相对表达水平。引物序列如表1所示。
1.2.2 Western blotting检测YTHDF1、dystrophin蛋白表达水平
提取肌肉组织总蛋白，使用BCA蛋白含量测定试剂盒进行蛋白定量。上样行SDS-PAGE电泳，并转移至PVDF膜上；加入5%脱脂奶粉封闭1 h，TBST清洗；加入YTHDF1、dystrophin抗体(1:1000)4 ℃孵育过夜；TBST清洗，加入辣根过氧化物酶标记的山羊抗鼠/兔IgG抗体(1:500)室温孵育1 h。使用ECL Plus化学发光试剂盒进行曝光，化学发光成像系统拍片，以GAPDH为内参照，ImageJ软件进行定量分析。
1.2.3 血清CPK含量检测
取血液样本，1000×g离心10 min，收集上清，按照CPK ELISA试剂盒说明书步骤检测血清CPK含量。
1.2.4 YTHDF1蛋白表达水平与血清CPK含量的相关性分析
采用Pearson相关性分析YTHDF1蛋白表达水平与血清CPK含量的相关性。
1.3 细胞培养
人类骨骼肌细胞系(SkMC)由中科院上海生命科学研究院细胞中心提供，置于含hEGF、地塞米松、L-谷氨酰胺和庆大霉素的DMEM培养基中，补充10% FBS，于37 °C、5% CO2条件下培养。
1.3.1 过表达载体构建及siRNA合成
根据NCBI GenBank数据库中YTHDF1编码区(CDS)序列，设计上下游分别含有Xho I和BamH I酶切位点的PCR引物扩增目的片段，双酶切、胶回收纯化后将其连接于进行相同双酶切处理的腺病毒表达载体，转化至大肠杆菌感受态细胞，过夜培养。第2天挑取单克隆摇菌，提取质粒后经Xho I和BamH I双酶切鉴定及测序后，确认载体构建成功。参照腺病毒包装规程进行病毒包装，并进行滴度检测。YTHDF1 siRNA及其阴性对照(NC siRNA)由广州锐博生物科技有限公司设计、合成。
1.3.2 细胞转染
取对数生长期SkMC细胞，接种于6孔板中(接种密度105个/cm2)。设置空载体组(转染对照空载体)、AAV-YTHDF1组(转染AAV-YTHDF1过表达载体)与Scrambled组(转染NC siRNA)、si-YTHDF1组(转染YTHDF1 siRNA)，待细胞融合至70%时，使用Lipofectamine 3000转染试剂进行转染，转染后于37 ℃培养48 h，收集细胞。
1.3.2.1 Western blotting检测YTHDF1过表达或敲低效率
提取各组SkMC细胞总蛋白，采用Western blotting检测YTHDF1蛋白表达水平，一抗YTHDF1抗体稀释比例为1∶1000。操作步骤同1.2.2。
1.3.2.2 EdU法检测细胞增殖情况
根据EdU试剂盒说明书步骤，使用5-乙基-2-脱氧尿苷(EdU)掺入法检测各组细胞增殖情况。加入EdU孵育细胞约2 h，4%多聚甲醛溶液固定30 min，0.5% Triton X-100孵育15 min，染色反应液孵育30 min。细胞核用Hoechst染色。随机选取5个区域于显微镜下观察拍片。
1.3.2.3 Western blotting检测dystrophin、YAP1、MyoG、MHC蛋白表达水平
提取SkMC细胞总蛋白，采用Western blotting检测dystrophin、YAP1、MyoG、MHC蛋白表达水平，操作步骤同1.2.2。一抗dystrophin抗体、YAP1抗体、MyoG抗体和MHC抗体稀释比例为1∶1000。
1.3.2.4 RT-qPCR检测YTHDF1、YAP1 mRNA表达水平
使用Trizol试剂提取SkMC细胞总RNA，采用RT-qPCR检测YTHDF1、YAP1 mRNA表达水平，操作步骤同1.2.1。
1.3.3 SRAMP在线数据库预测
采用SRAMP在线数据库预测YAP1 mRNA序列中的m6A修饰位点。
1.3.4 RNA免疫沉淀反应(RIP)检测YTHDF1与YAP1 mRNA的结合情况
根据Magna RIP RNA结合蛋白免疫沉淀试剂盒说明书步骤进行RIP测定。用补充有蛋白酶和RNase抑制剂的RNA裂解缓冲液裂解SkMC细胞，并在4 ℃下用涂有anti-YTHDF1抗体、anti-m6A抗体或IgG的蛋白A/G磁珠培养细胞裂解物过夜。洗涤后，纯化免疫沉淀RNA，然后采用RT-qPCR进行定量分析。
1.3.5 mRNA稳定性检测
转染YTHDF1 siRNA 16 h后，使用转录抑制剂放线菌素D干预，收取干预0、2、4、8及16 h的细胞，提取总RNA，用酶标仪检测RNA浓度，反转录得到cDNA，通过RT-qPCR检测YAP1 mRNA表达水平，分析降解速率。
1.4 动物实验
30只雄性SPF清洁级Mdx小鼠，4周龄，体重10~18 g，购自西安交通大学实验动物中心[实验动物许可证号：SYXK(陕)2016-005]，饲养于12 h光照/12 h黑暗交替、温度(22±3) ℃、湿度45%~60%环境下，自由摄食饮水。本动物实验经西安市儿童医院实验动物福利与伦理委员会批准(20160918-1F)。
1.4.1 DMD小鼠模型构建
将30只Mdx小鼠随机分为空载体组与AAV-YTHDF1组，每组15只。空载体组腹腔注射200 μl空载体，AAV-YTHDF1组腹腔注射200 μl AVV-YTHDF1。注射时间为第1天、第1次注射后48 h、第1次注射后15 d。每天监测小鼠的饮食、呼吸、精神状态和体重。距第1次注射20周处死小鼠，采集肌肉组织和器官，检测肌肉组织和器官湿重、纤维直径及纤维类型。
1.4.2 Western blotting检测YTHDF1、dystrophin蛋白表达水平
将肌肉组织制备为组织匀浆液，采用Western blotting检测YTHDF1、dystrophin蛋白表达水平，具体操作同1.2.2。
1.4.3 HE染色
小鼠处死后立即取后肢腓肠肌和股四头肌，组织块大小不超过0.5 cm3。用0.9%生理盐水漂洗血液与污渍，将组织放入包埋盒中，迅速浸泡于4%多聚甲醛溶液中固定，常规切片后行HE染色，于显微镜下观察。
1.5 统计学处理
采用SPSS 22.0软件进行统计分析。所有实验数据以$\bar{x}±s$表示，两组间比较采用t检验，多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。P<0.05为差异有统计学意义。
2 结果
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2.1 YTHDF1在DMD患者肌肉组织中的表达情况
RT-qPCR和Western blotting检测结果显示，与对照组比较，DMD组患者肌肉组织中YTHDF1 mRNA和蛋白表达水平均明显降低(P<0.01或P<0.001，图1A、B)。ELISA检测结果显示，与对照组比较，DMD组患者血清CPK含量明显增加(P<0.001，图1C)。Pearson相关分析结果显示，DMD患者YTHDF1蛋白表达水平与血清CPK含量呈负相关(r2=0.8658，P<0.0001，图1D)。Western blotting检测结果显示，与对照组比较，DMD组患者肌肉组织中dystrophin蛋白表达水平明显降低(P<0.01，图1E)。
2.2 过表达YTHDF1对肌细胞增殖和dystrophin表达的影响
Western blotting检测结果显示，与空载体组比较，AAV-YTHDF1组SkMC细胞中YTHDF1蛋白表达水平明显升高(P<0.01，图2A)，表明YTHDF1过表达实验成功。与空载体组比较，AAV-YTHDF1组SkMC细胞中dystrophin、MyoG和MHC蛋白表达水平明显升高(P<0.01，图2B)。EdU检测结果显示，与空载体组比较，AAV-YTHDF1组EdU阳性细胞率明显增高(P<0.01，图2C)。
2.3 敲低YTHDF1对肌细胞增殖和dystrophin表达的影响
Western blotting检测结果显示，与Scrambled组比较，si-YTHDF1组SkMC细胞中YTHDF1蛋白表达水平明显降低(P<0.01，图3A)，表明YTHDF1敲低实验成功。与Scrambled组比较，si-YTHDF1组SkMC细胞中dystrophin、MyoG和MHC蛋白表达水平明显降低(P<0.01，图3B)。EdU检测结果显示，与Scrambled组比较，si-YTHDF1组EdU阳性细胞率明显降低(P<0.05，图3C)。
2.4 YTHDF1与YAP1 mRNA的相互作用
SRAMP在线数据库预测结果显示，YAP1 mRNA上存在多个m6A修饰位点(图4A)；RIP检测结果显示，Anti-YTHDF1抗体组中YAP1 mRNA富集，而Anti-YTHDF1抗体组与IgG抗体组中dystrophin mRNA含量无明显差异(图4B)；anti-m6A抗体组中YAP1 mRNA富集(图4C)，表明YTHDF1通过识别YAP1 mRNA的m6A修饰与YAP1 mRNA相互作用。
2.5 YTHDF1对YAP1表达和YAP1 mRNA稳定性的影响
Western blotting检测结果显示，与空载体组比较，AAV-YTHDF1组YAP1蛋白表达水平明显升高(P<0.01)；与Scrambled组比较，si-YTHDF1组YAP1蛋白表达水平明显降低(P<0.01，图5A)。放线菌素D干预实验结果显示，与空载体组比较，AAV-YTHDF1组YAP1 mRNA半衰期延长，同一时间点YAP1 mRNA降解减少(P<0.001，图5B)。
2.6 YTHDF1对DMD小鼠肌肉生长和肌肉质量的影响
AAV-YTHDF1组与空载体组小鼠初次注射时体重无明显差异(P>0.05)，但20周时，AAV-YTHDF1组小鼠体重较空载体组增加，差异有统计学意义(P<0.01，图6A)。Western blotting检测结果显示，与空载体组比较，AAV-YTHDF1组小鼠肌肉组织中YTHDF1和dystrophin蛋白表达水平均明显升高
(P<0.01，图6B)。与空载体组比较，AAV-YTHDF1组小鼠腓肠肌、股四头肌、三头肌肌肉重量增加(P<0.05)；但两组腹股沟、性腺或腹膜后脂肪垫重量无明显差异(P>0.05，图6C)。HE染色结果显示，与空载体组比较，AAV-YTHDF1组小鼠炎性细胞浸润减少，坏死面积缩小，组织病理学改变明显得到改善(P<0.05)；此外，与空载体组比较，AAV-YTHDF1组小鼠腓肠肌和股四头肌纤维面积明显增大(P<0.05，图6D)。
3 讨论
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DMD是最常见的遗传性神经性肌肉病，起病隐匿且预后差，大多数患者早期无明显临床症状，导致诊断困难和误诊。在美国，DMD从症状开始到确诊约有2.5年的延迟，而在全球其他地区，延迟时间则更长[8]。DMD是一种主要影响男孩的疾病，其特征是进行性肌肉退行性变和萎缩，导致心肌病和呼吸衰竭，最终导致患儿过早死亡[9]。
DMD的分子遗传学基础是dystrophin基因突变[10]。Dystrophin基因突变导致dystrophin蛋白突变，而dystrophin位于肌细胞膜内侧面，是一种大型支架蛋白，可与相关糖蛋白组成DAPC[10-11]。DAPC将细胞外基质锚定到细胞骨架上，使肌肉纤维抵抗损伤的能力明显增强，是肌肉收缩过程中维持肌膜结构完整性的关键蛋白[12]。虽然多数骨骼肌的初始形成在发育过程中不受阻碍，但由于与肌肉收缩相关的机械应力导致外周膜受损，肌肉纤维会随着个体的成熟而迅速退化[13]。随着疾病的加重，被称为卫星细胞的骨骼肌祖细胞无法充分增殖和分化以替代受损的肌肉纤维[14]。有报道显示，可以利用内源性机制促进肌细胞持续增殖来维持骨骼肌的再生能力，减轻DMD造成的损害[15]。
Dystroglycan 1又称为dystrophin相关糖蛋白，通常与dystrophin形成蛋白复合物发挥作用。Dystroglycan 1可直接与Hippo通路的效应因子YAP1结合，抑制小鼠心肌细胞的增殖，当编码dystroglycan 1的基因被敲除时，这种相互作用被破坏，而Hippo诱导的YAP1磷酸化增强了YAP1与dystroglycan 1的相互作用[12]。YAP1作为Hippo信号通路下游的关键效应因子，可调节细胞分化、增殖和凋亡，控制器官发育和再生，以及维持正常组织的稳态，在多种生理病理过程中发挥重要作用[16]。有研究发现，dystrophin与Hippo信号通路的关键激酶Wts存在相互作用，并且dystrophin能够通过结合Wts调控果蝇的器官发育[17]。本研究发现，DMD患者肌肉组织中dystrophin表达水平明显降低；过表达YTHDF1可促进肌细胞增殖，使dystrophin蛋白表达水平升高；敲低YTHDF1可抑制肌细胞增殖，使dystrophin蛋白表达水平降低。
YAP1作为潜在的致癌基因[18]，目前研究主要集中于YAP1上下游分子的筛选[19]；然而，关于YAP1表达水平调控的机制尚未完全清楚。m6A修饰是真核细胞中最丰富的RNA内部修饰，可被m6A结合蛋白(reader)识别，包括YTHDF1-3、IGF2BP1-3和YTHDC1-2[20]。本研究发现，与正常肌肉组织比较，m6A识别蛋白YTHDF1在DMD肌肉组织中表达降低。既往研究发现，YTHDFs可识别YAP1 mRNA的m6A修饰，其可通过与m6A修饰的mRNA结合来提高稳定性，从而促进YAP1的表达[21-24]。本研究结果证实了这一现象，即过表达YTHDF1通过延长YAP1 mRNA的半衰期来稳定YAP1 mRNA表达。
MyoG和MHC是成肌细胞分化过程中骨骼肌特异性表达的关键基因。MyoG是骨骼肌分化的决定因子，调控成肌细胞融合和肌纤维形成[25]，MyoG敲除小鼠因无肌肉形成，在胚胎期死亡[26]；MHC是构成骨骼肌纤维内粗肌丝的主要成分。肌纤维类型主要由肌纤维内表达的MHC类型决定[27]。本研究发现，过表达YTHDF1可促进MyoG和MHC蛋白的表达；敲低YTHDF1可抑制MyoG和MHC蛋白的表达。
综上所述，本研究结果表明，YTHDF1通过识别YAP1 mRNA的m6A修饰促进YAP1 mRNA的稳定性，进而促进YAP1/dystrophin介导的肌细胞增殖，改善DMD小鼠的肌肉萎缩。未来可进一步完善DMD肌肉萎缩的分子调控网络，以为DMD的诊断及治疗提供理论依据和新的视角。
基金
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  • 陕西省社会发展科技攻关项目(2015SF214)
  • 西安市儿童医院院级科研项目(2022G03)
文献
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2023年第48卷第12期
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doi: 10.11855/j.issn.0577-7402.1795.2023.0614
  • 接收时间：2022-08-26
  • 首发时间：2025-11-24
  • 出版时间：2023-12-28
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  • 收稿日期：2022-08-26
  • 录用日期：2022-11-05
基金
Shaanxi Science and Technology Research Project for Social Development(2015SF214)
陕西省社会发展科技攻关项目(2015SF214)
Scientific Research Project of Xi'an Children's Hospital(2022G03)
西安市儿童医院院级科研项目(2022G03)
作者信息
    西安市儿童医院神经内科，陕西西安 710002

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