NOS2基因DNA甲基化编辑调节NO浓度影响肌肉发育通路基因的表达

doi:10.11843/j.issn.0366-6964.2024.03.012

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (3): 984-994.doi: 10.11843/j.issn.0366-6964.2024.03.012

NOS2基因DNA甲基化编辑调节NO浓度影响肌肉发育通路基因的表达

申琦^1,2,3, 王凯⁴, 赵真坚^1,2,3, 陈栋^1,2,3, 余杨^1,2,3, 崔晟頔^1,2,3, 王俊戈^1,2,3, 陈子旸^1,2,3, 吴平先⁵, 唐国庆^1,2,3*

1. 四川农业大学动物科技学院农业农村部畜禽生物组学重点实验室, 成都 611130;
2. 四川农业大学畜禽遗传资源发掘与创新利用四川省重点实验室, 成都 611130;
3. 四川农业大学动物科技学院猪禽种业全国重点实验室, 成都 611130;
4. 新希望六和股份有限公司, 北京 100102;
5. 国家生猪技术创新中心, 重庆 402460

收稿日期:2023-09-01 出版日期:2024-03-23 发布日期:2024-03-27
通讯作者: 唐国庆,主要从事猪遗传育种研究,E-mail:tyq003@163.com
作者简介:申琦(1997-),女,四川苍溪人,硕士生,主要从事动物遗传育种与繁殖研究,E-mail:2021202042@stu.sicau.edu.cn
基金资助:
国家生猪技术创新中心先导科技项目（NCTIP-XD/B01）；四川省科技厅项目（2020YFN0024；2021ZDZX0008；2021YFYZ0030）；四川省猪创新团队项目（sccxtd-2022-08）

Regulation of NO Concentration by NOS2 Gene DNA Methylation Editing Affects the Expression of Muscle Development Pathway Genes

SHEN Qi^1,2,3, WANG Kai⁴, ZHAO Zhenjian^1,2,3, CHEN Dong^1,2,3, YU Yang^1,2,3, CUI Shengdi^1,2,3, WANG Junge^1,2,3, CHEN Ziyang^1,2,3, WU Pingxian⁵, TANG Guoqing^1,2,3*

1. Key Laboratory of Livestock and Poultry Multi-omics of Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China;
2. Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China;
3. State Key Laboratory of Swine and Poultry Breeding Industry, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China;
4. New Hope Liuhe Co. Ltd., Beijing 100102, China;
5. National Center of Technology Innovation for Pigs, Chongqing 402460, China

Received:2023-09-01 Online:2024-03-23 Published:2024-03-27

摘要/Abstract

摘要： 旨在探究对一氧化氮合成酶（NOS）2进行DNA甲基化编辑后对肌红蛋白的调控作用机制，并研究其对神经元一氧化氮合酶（nNOS）和内皮细胞一氧化氮合酶（eNOS）的表达水平是否有影响。本试验采用DNA甲基化编辑技术在猪肾细胞系PK-15细胞对基因NOS2进行甲基化编辑，在猪肾细胞中检测NOS2基因启动子DNA甲基化水平、NOS2表达水平、细胞内一氧化氮浓度以及肌纤维和肌红蛋白相关基因的表达。结果发现，NOS2基因启动子前500 bp区域平均DNA甲基化水平降低，但差异不显著，而gRNA2结合位点附近的CpG位点甲基化水平增高。此外，对NOS2进行甲基化编辑后，NOS2基因表达量和其iNOS蛋白质表达量显著降低。随着NOS2表达降低，nNOS和eNOS表达显著增加。MYHC-Ⅱb和MYHC-Ⅱx的表达水平显著下降，Myoglobin蛋白表达也显著下降。综上，NOS2基因的异常表达能够影响细胞中的NO生成，同时也会影响NOS1和NOS3的表达，以及和肌肉发育相关基因的表达。

关键词: NOS2, DNA甲基化, NO, 肌纤维, 肌红蛋白

Abstract: This study aimed to explore the regulation mechanism of DNA methylation editing of nitric oxide synthase (NOS) 2 on Myoglobin, and study its effect on the expression level of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS). In this experiment, DNA methylation editing technology was used to methylate NOS2 in porcine kidney cell line PK-15 cells, and the DNA methylation level of the NOS2 gene promoter, the expression level of NOS2, the concentration of intracellular nitric oxide, as well as the expression level of myofibrillar-and myoglobin-related genes were detected in porcine kidney cells. The results revealed that the average DNA methylation level of the first 500 bp region of the NOS2 gene promoter was reduced, but the difference was not significant, while the methylation level of the CpG site near the gRNA2 binding site was increased. In addition, after DNA methylation editing of NOS2 gene, NOS2 gene expression and its iNOS protein expression were significantly reduced. With the decrease of NOS2 expression, nNOS and eNOS expression increased significantly, the expression level of MYHC-Ⅱb and MYHC-Ⅱx decreased significantly, and the expression of Myoglobin protein decreased significantly. In conclusion, the abnormal expression of the NOS2 gene can affect the generation of NO in cells, as well as the expression of NOS1 and NOS3, and the expression of genes related to muscle development.

Key words: NOS2, DNA methylation, NO, muscle fiber, myoglobin

中图分类号:

S828.2

申琦, 王凯, 赵真坚, 陈栋, 余杨, 崔晟頔, 王俊戈, 陈子旸, 吴平先, 唐国庆. NOS2基因DNA甲基化编辑调节NO浓度影响肌肉发育通路基因的表达[J]. 畜牧兽医学报, 2024, 55(3): 984-994.

SHEN Qi, WANG Kai, ZHAO Zhenjian, CHEN Dong, YU Yang, CUI Shengdi, WANG Junge, CHEN Ziyang, WU Pingxian, TANG Guoqing. Regulation of NO Concentration by NOS2 Gene DNA Methylation Editing Affects the Expression of Muscle Development Pathway Genes[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 984-994.

参考文献

[1] ZHANG L, ZHENG Y L, WANG R, et al.Exercise for osteoporosis:a literature review of pathology and mechanism[J].Front Immunol, 2022, 13:1005665.
[2] SAVIANA M, LE P, MICALO L, et al.Crosstalk between miRNAs and DNA methylation in cancer[J].Genes (Basel), 2023, 14(5):1075.
[3] MENG H, CAO Y, QIN J Z, et al.DNA methylation, its mediators and genome integrity[J].Int J Biol Sci, 2015, 11(5):604-617.
[4] MCGEE S L, HARGREAVES M.Epigenetics and exercise[J].Trends Endocrinol Metab, 2019, 30(9):636-645.
[5] LING C, RÖNN T.Epigenetics in human obesity and type 2 diabetes[J].Cell Metab, 2019, 29(5):1028-1044.
[6] SAYOLS-BAIXERAS S, SUBIRANA I, FERNÁNDEZ-SANLÉS A, et al.DNA methylation and obesity traits:an epigenome-wide association study.The REGICOR study[J].Epigenetics, 2017, 12(10):909-916.
[7] BAGHERIPOUR F, JEDDI S, KASHFI K, et al.Metabolic effects of L-citrulline in type 2 diabetes[J].Acta Physiol (Oxf), 2023, 237(3):e13937.
[8] TEKWE C D, LUAN Y Y, MEININGER C J, et al.Dietary supplementation with L-leucine reduces nitric oxide synthesis by endothelial cells of rats[J].Exp Biol Med (Maywood), 2023, 248(18):1537-1549.
[9] CHEN P Y, FENG S H, JOO J W J, et al.A comparative analysis of DNA methylation across human embryonic stem cell lines[J].Genome Biol, 2011, 12(7):R62.
[10] NERY DA SILVA A, SILVA ARAUJO M, PÉRTILLE F, et al.How epigenetics can enhance pig welfare?[J]. Animals (Basel), 2022, 12(1):32.
[11] ZHAO Y X, HOU Y, XU Y Y, et al.A compendium and comparative epigenomics analysis of cis-regulatory elements in the pig genome[J].Nat Commun, 2021, 12(1):2217.
[12] THOMPSON R P, NILSSON E, SKINNER M K.Environmental epigenetics and epigenetic inheritance in domestic farm animals[J].Anim Reprod Sci, 2020, 220:106316.
[13] MATTILA J T, THOMAS A C.Nitric oxide synthase:non-canonical expression patterns[J].Front Immunol, 2014, 5:478.
[14] HALL D T, MA J F, DI MARCO S, et al.Inducible nitric oxide synthase (iNOS) in muscle wasting syndrome, sarcopenia, and cachexia[J].Aging (Albany NY), 2011, 3(8):702-715.
[15] MACCALLINI C, AMOROSO R.Preface to nitric oxide modulators in health and disease I[J].Molecules, 2022, 27(20):6820.
[16] ALMANNAI M, EL-HATTAB A W.Nitric oxide deficiency in mitochondrial disorders:the utility of arginine and citrulline[J].Front Mol Neurosci, 2021, 14:682780.
[17] NATH P, MAITRA S.Physiological relevance of nitric oxide in ovarian functions:an overview[J].Gen Comp Endocrinol, 2019, 279:35-44.
[18] HE W X, ZHOU H F, HE X Y.Aloperine protects beta-cells against streptozocin-induced injury to attenuate diabetes by targeting NOS1[J].Eur J Pharmacol, 2022, 916:174721.
[19] KRÓL M, KEPINSKA M.Human nitric oxide synthase-its functions, polymorphisms, and inhibitors in the context of inflammation, diabetes and cardiovascular diseases[J].Int J Mol Sci, 2021, 22(1):56.
[20] PAUTZ A, LI H G, KLEINERT H.Regulation of NOS expression in vascular diseases[J].Front Biosci (Landmark Ed), 2021, 26(5):85-101.
[21] PAPPAS G, WILKINSON M L, GOW A J.Nitric oxide regulation of cellular metabolism:adaptive tuning of cellular energy[J].Nitric Oxide, 2023, 131:8-17.
[22] ROY R, WILCOX J, WEBB A J, et al.Dysfunctional and dysregulated nitric oxide synthases in cardiovascular disease:mechanisms and therapeutic potential[J].Int J Mol Sci, 2023, 24(20):15200.
[23] ALMAZROUE H, JIN Y, NELIN L D, et al.Human pulmonary microvascular endothelial cell DDAH1-mediated nitric oxide production promotes pulmonary smooth muscle cell apoptosis in co-culture[J].Am J Physiol Lung Cell Mol Physiol, 2023, 325(3):L360-L367.
[24] BOO Y C, JO H.Flow-dependent regulation of endothelial nitric oxide synthase:role of protein kinases[J].Am J Physiol Cell Physiol, 2003, 285(3):C499-C508.
[25] MANCINI R A, HUNT M C.Current research in meat color[J].Meat Sci, 2005, 71(1):100-121.
[26] BROWN G C, BORUTAITE V.Nitric oxide and mitochondrial respiration in the heart[J].Cardiovasc Res, 2007, 75(2):283-290.
[27] VENHOFF N, LEBRECHT D, PFEIFER D, et al.Muscle-fiber transdifferentiation in an experimental model of respiratory chain myopathy[J].Arthritis Res Ther, 2012, 14(5):R233.
[28] SESSA W C.The nitric oxide synthase family of proteins[J].J Vasc Res, 1994, 31(3):131-143.
[29] MOSKALEVA P V, SHNAYDER N A, PETROVA M M, et al.The role of single nucleotide variants of NOS1, NOS2, and NOS3 genes in the development of the phenotype of migraine and arterial hypertension[J]. Brain Sci, 2021, 11(6):753.
[30] SEARLES NIELSEN S, CHECKOWAY H, CRISWELL S R, et al.Inducible nitric oxide synthase gene methylation and parkinsonism in manganese-exposed welders[J].Parkinsonism Relat Disord, 2015, 21(4):355-360.
[31] HATTORI Y, HATTORI S, KASAI K.Lipopolysaccharide activates Akt in vascular smooth muscle cells resulting in induction of inducible nitric oxide synthase through nuclear factor-kappa B activation[J].Eur J Pharmacol, 2003, 481(2-3):153-158.
[32] SILVA G B, GARVIN J L.Akt1 mediates purinergic-dependent NOS3 activation in thick ascending limbs[J].Am J Physiol Renal Physiol, 2009, 297(3):F646-F652.
[33] MACMICKING J, XIE Q W, NATHAN C.Nitric oxide and macrophage function[J].Annu Rev Immunol, 1997, 15:323-350.
[34] REISBERG P, OLSON J S, PALMER G.Kinetic resolution of ligand binding to the α and β chains within human hemoglobin[J].J Biol Chem, 1976, 251(14):4379-4383.
[35] RAYNER B S, HUA S S, SABARETNAM T, et al.Nitric oxide stimulates myoglobin gene and protein expression in vascular smooth muscle[J].Biochem J, 2009, 423(2):169-177.
[36] VANEK T, KOHLI A.Biochemistry, myoglobin[M]//StatPearls [Internet].Treasure Island (FL):StatPearls Publishing, 2023.
[37] CHANG K C, FERNANDES K.Developmental expression and 5' cloning of the porcine 2x and 2b myosin heavy chain genes[J].DNA Cell Biol, 1997, 16(12):1429-1437.
[38] RYU Y C, KIM B C.The relationship between muscle fiber characteristics, postmortem metabolic rate, and meat quality of pig longissimus dorsi muscle[J].Meat Sci, 2005, 71(2):351-357.
[39] KONE B C, KUNCEWICZ T, ZHANG W Z, et al.Protein interactions with nitric oxide synthases:controlling the right time, the right place, and the right amount of nitric oxide[J].Am J Physiol Renal Physiol, 2003, 285(2):F178-F190.
[40] SHULTZ P J, TOLINS J P.Adaptation to increased dietary salt intake in the rat.Role of endogenous nitric oxide[J].J Clin Invest, 1993, 91(2):642-650.
[41] WELCH W J, WILCOX C S.Macula densa arginine delivery and uptake in the rat regulates glomerular capillary pressure.Effects of salt intake[J].J Clin Invest, 1997, 100(9):2235-2242.

NOS2基因DNA甲基化编辑调节NO浓度影响肌肉发育通路基因的表达

Regulation of NO Concentration by NOS2 Gene DNA Methylation Editing Affects the Expression of Muscle Development Pathway Genes

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	王海波, 占今舜, 谷志勇, 陈新锋, 潘月, 贾浩滨, 钟小军, 李开嵘, 赵生国, 霍俊宏. 湖羊与三元杂交绵羊夏杜湖、夏澳湖肉质特性比较研究[J]. 畜牧兽医学报, 2024, 55(1): 110-119.
[2]	金美林, 李桃桃, 孙东晓, 魏彩虹. 表观遗传调控在畜禽脂肪沉积机制中的研究进展[J]. 畜牧兽医学报, 2023, 54(3): 855-867.
[3]	杨小耿, 张慧珠, 李键, 向华, 何翃闳. DNA甲基化在哺乳动物卵母细胞和早期胚胎发育中的研究进展[J]. 畜牧兽医学报, 2023, 54(2): 443-450.
[4]	庞立川, 单艳菊, 刘一帆, 章明, 甘达峰, 屠云洁, 姬改革, 巨晓军, 束婧婷, 邹剑敏. METTL16在鸡不同类型肌肉中的表达规律及其对肌肉功能的调控作用[J]. 畜牧兽医学报, 2023, 54(2): 545-553.
[5]	贾紫洁, 图格琴, 丁文淇, 任秀娟, 刘慧莹, 李欣泽, 翠芳, 芒来, 白东义. 蒙古马全身主要骨骼肌表型谱的构建及比较研究[J]. 畜牧兽医学报, 2023, 54(2): 596-607.
[6]	杨昊天, 陈永平, 王志强, 黄宇翔, 马志刚, 邹跃, 魏念冬, 张红, 李鑫, 董佳强, 吕明哲, 李洪彬, 刘力威, 杨昊轩, 张国华, 刘雪松, 钟鹏, 石荷叶, 寇玉红, 陈志峰. 右美托咪定抑制NOX4缓解急性应激致大鼠肾损伤[J]. 畜牧兽医学报, 2023, 54(2): 779-786.
[7]	张宸艺博, 余彤, 任斌斌, 郑睿智, 朱文治, 苏建民. 动物早期胚胎发育中表观重编程的机制[J]. 畜牧兽医学报, 2023, 54(12): 4898-4909.
[8]	马帅, 王燕, 庄新娟, 王文正, 赵茹茜. 母鹅日粮添加甜菜碱通过IGFs信号通路促进子代胸肌肌纤维肥大[J]. 畜牧兽医学报, 2023, 54(12): 5112-5124.
[9]	赵亚亚, 常江, 剧柠, 罗玉龙. 日粮中添加植物多酚调控肉色稳定性的作用机制[J]. 畜牧兽医学报, 2023, 54(1): 36-47.
[10]	陈香凝, 刘萌萌. 猫肥厚型心肌病与心肌纤维化[J]. 畜牧兽医学报, 2023, 54(1): 80-87.
[11]	叶状, 王乐乐, 汪飞燕, 刘悦, 彭月梅, 宿世杰, 候照峰, 许金俊, 陶建平, 刘丹丹. 毒害艾美耳球虫氧化还原酶EnOXIO1的原核表达与定位分析[J]. 畜牧兽医学报, 2022, 53(5): 1553-1561.
[12]	张志恒, 白荟, 彭立颖, 张思学, 贾冰, 魏成威, 高利. 赛拉嗪-咪达唑仑复合麻醉剂对山羊大脑和海马NO/cGMP信号转导系统及神经递质的影响[J]. 畜牧兽医学报, 2022, 53(4): 1289-1297.
[13]	周力, 高占红, 侯生珍, 杨葆春, 王志有, 桂林生. 初生和成年黑藏羊肉品质与肌纤维特性差异分析[J]. 畜牧兽医学报, 2022, 53(3): 700-710.
[14]	邹荣华, 吴晓妮, 陈启伟, 宫晓炜, 王燕萍, 郑福英, 储岳峰. Enolase在鸭疫里默氏杆菌侵袭鸭脑微血管内皮细胞中的作用[J]. 畜牧兽医学报, 2022, 53(12): 4389-4397.
[15]	侯任达, 张润, 侯欣华, 王立贤, 张龙超. 畜禽肌纤维发育规律及相关基因研究进展[J]. 畜牧兽医学报, 2022, 53(10): 3279-3286.