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
- 解放军医学杂志 2023年48卷第12期 页码：1403-1411
- 作者机构：
西安市儿童医院神经内科，陕西西安 710002
- 作者简介：
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
李蓓，E-mail：wxvjd9pq@163.com
- 基金信息：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)
DOI：10.11855/j.issn.0577-7402.1795.2023.0614
中图分类号：
扫 描 看 全 文
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
references
Blau HM, Webster C, Pavlath GK. Defective myoblasts identified in Duchenne muscular dystrophy[J]. Proc Natl Acad Sci U S A, 1983, 80(15): 4856-4860.
Li S, Xu ZY, Yu ZH, et al. Effect of semaphorin 3A on myoblast function and its transcriptional regulation mechanism[J]. Med J Chin PLA, 2022, 47(7): 701-708.
李上, 徐子瑛, 于子惠, 等. 信号蛋白3A对成肌细胞功能的影响及其转录调控机制[J]. 解放军医学杂志, 2022, 47(7): 701-708.
Hoffman EP, JrBrown RH, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus[J]. Cell, 1987, 51(6): 919-928.
Wang YM, Tian GL, Ji W, et al. Application of creatine kinase isoenzyme MM in screening neonatal Duchenne muscular dystrophy[J]. Lab Med, 2020, 35(11): 1115-1119.
王燕敏, 田国力, 纪伟, 等. 肌酸激酶MM同工酶在新生儿杜氏肌营养不良症筛查中的应用[J]. 检验医学, 2020, 35(11): 1115-1119.
Liang Y, Han H, Xiong Q, et al. METTL3-mediated m6A methylation regulates muscle stem cells and muscle regeneration by notch signaling pathway[J]. Stem Cells Int, 2021, 2021: 9955691.
Yu B, Liu J, Zhang J, et al. Regulatory role of RNA N6-methyladenosine modifications during skeletal muscle development[J]. Front Cell Dev Biol, 2022, 10: 929183.
Yasutake H, Lee JK, Hashimoto A, et al. Decreased YAP activity reduces proliferative ability in human induced pluripotent stem cell of Duchenne muscular dystrophy derived cardiomyocytes[J]. Sci Rep, 2021, 11(1): 10351.
Wong SH, McClaren BJ, Archibald AD, et al. A mixed methods study of age at diagnosis and diagnostic odyssey for Duchenne muscular dystrophy[J]. Eur J Hum Genet, 2015, 23(10): 1294-1300.
Amoasii L, Hildyard JCW, Li H, et al. Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy[J]. Science, 2018, 362(6410): 86-91.
Blake DJ, Weir A, Newey SE, et al. Function and genetics of dystrophin and dystrophin-related proteins in muscle[J]. Physiol Rev, 82(2): 291-329.
Wein N, Alfano L,Flanigan KM. Genetics and emerging treatments for Duchenne and becker muscular dystrophy[J]. Pediatr Clin North Am, 2015, 62(3): 723-742.
Morikawa Y, Heallen T, Leach J, et al. Dystrophin-glycoprotein complex sequesters Yap to inhibit cardiomyocyte proliferation[J]. Nature, 2017, 547(7662): 227-231.
Guiraud S, Aartsma-Rus A, Vieira NM, et al. The pathogenesis and therapy of muscular dystrophies[J]. Annu Rev Genomics Hum Genet, 2015, 16: 281-308.
Almada AE, Wagers AJ. Molecular circuitry of stem cell fate in skeletal muscle regeneration, ageing and disease[J]. Nat Rev Mol Cell Biol, 2016, 17(5): 267-279.
Chang NC, Chevalier FP, Rudnicki MA. Satellite cells in muscular dystrophy-lost in polarity[J]. Trends Mol Med, 2016, 22(6): 479-496.
Shibata M, Ham K, Hoque MO. A time for YAP1: tumorigenesis, immunosuppression and targeted therapy[J]. Int J Cancer, 2018, 143(9): 2133-2144.
Zhang XT, Wu SA. Dystrophins are involved in the regulation of the Hippo signaling pathway[J]. Acta Sci Nat Univer Nankai (Natur Sci Edit), 2021, 54(3): 41-47.
张晓亭, 吴世安. 肌营养不良蛋白dys参与调控Hippo信号通路[J]. 南开大学学报(自然科学版), 2021, 54(3): 41-47.
Song S, Honjo S, Jin J, et al. The Hippo coactivator YAP1 mediates EGFR overexpression and confers chemoresistance in esophageal cancer[J]. Clin Cancer Res, 2015, 21(11): 2580-2590.
Jin D, Guo J, Wu Y, et al. m6A mRNA methylation initiated by METTL3 directly promotes YAP translation and increases YAP activity by regulating the MALAT1-miR-1914-3p-YAP axis to induce NSCLC drug resistance and metastasis[J]. J Hematol Oncol, 2021, 14(1): 32.
Zhang S. Mechanism of N6-methyladenosine modification and its emerging role in cancer[J]. Pharmacol Ther, 2018, 189: 173-183.
Jin D, Guo J, Wu Y, et al. m6A demethylase ALKBH5 inhibits tumor growth and metastasis by reducing YTHDFs-mediated YAP expression and inhibiting miR-107/LATS2-mediated YAP activity in NSCLC[J]. Mol Cancer, 2020, 19(1): 40.
Zhuang M, Li X, Zhu J, et al. The m6A reader YTHDF1 regulates axon guidance through translational control of Robo3.1 expression[J]. Nucleic Acids Res, 2019, 47(9): 4765-4777.
Du H, Zhao Y, He J, et al. YTHDF2 destabilizes m6A-containing RNA through direct recruitment of the CCR4-NOT deadenylase complex[J]. Nat Commun, 2016, 7: 12626.
Shi H, Wang X, Lu Z, et al. YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA[J]. Cell Res, 2017, 27(3): 315-328.
Buckingham M, Rigby PWJ. Gene regulatory networks and transcriptional mechanisms that control myogenesis[J]. Dev Cell, 2014, 28(3): 225-238.
Nabeshima Y, Hanaoka K, Hayasaka M, et al. Myogenin gene disruption results in perinatal lethality because of severe muscle defect[J]. Nature, 1993, 364(6437): 532-535.
Zhang J, Wang SJ, Liu Y, et al. Knockdown of RACK1 inhibits the expression of MyoG and MHC in C2C12 cells[J]. Chin J Animal Sci, 2017, 53(3): 106-111.
张晶, 王胜军, 刘妍, 等. 敲低RACK1基因抑制C2C12细胞中MyoG和MHC基因表达[J]. 中国畜牧杂志, 2017, 53(3): 106-111.
0
浏览量
0
下载量
0
CSCD
0
CNKI被引量
- 网络PDF下载
- Meta-XML下载
- BibTeX下载
- Endnote下载
- RefMan 下载
- NoteExpress下载
- NoteFirst下载
关联资源
相关文章
相关作者
相关机构
- 技术支持由北京北大方正电子有限公司提供 京ICP备09064830号-19
京公网安备11010802024621 - 本系统建议在Chrome、 IE9+ 以上版本浏览器阅读本站内容，360浏览器请切换至极速模式
- Cookies帮助我们提供服务并提供个性化体验。使用本网站，即表示您同意我们使用Cookies