5-Aza-dC对牛成肌细胞MyoD1启动子甲基化及mRNA表达的影响

doi:10.11843/j.issn.0366-6964.2021.09.007

Abstract

Abstract: The aim of this study was to investigate the effects of 5-Aza-2'-deoxycytidine (5-Aza-dC) on the proliferation of bovine myoblasts, promoter methylation and mRNA expression of myogenic differentiation 1 (MyoD1). In this study, the Guanling cattle myoblasts cryopreserved in the early stage were recovered and cultured. After they had grown to the logarithmic growth phase, the bovine myoblasts were treated by different concentrations of 5-Aza-dC. The flow cytometry was used to detect cell apoptosis and cycle. The methylation level of MyoD1 promoter was detected by high-throughput detection methods, the expression level of MyoD1 and related genes were detected by qRT-PCR. The results showed that 0.1 μmol·L^-1 5-Aza-dC was the optimal concentration, and the expression of anti-apoptotic factor Bcl was significantly decreased (P<0.01), the expression of pro-apoptotic factor Bax was significantly increased (P<0.01), and the expression of pro-apoptotic factor Caspase-9 was significantly increased (P<0.05); The expression of Cyclin A2 was significantly increased (P<0.05), but the expression of Cyclin B1 and Cyclin D were not significantly affected. The MyoD1 promoter methylation level in blank group and experimental group was detected, and the MyoD1 promoter methylation level in experimental group was significantly lower than that in blank group (P<0.01), while the mRNA expression level was significantly higher in experimental group than that in blank group (P<0.01). In summary, 5-Aza-dC can regulate the proliferation and apoptosis of Guanling cattle myoblasts by changing the expressions of Caspase-9, Bcl, Bax, Cyclin A2 and other genes, and can effectively reduce the methylation level of MyoD1 gene promoter (P<0.01), and significantly increase its mRNA expression (P<0.01). Low concentration of 5-Aza-dC can promote cell apoptosis and regulate MyoD1 methylation level to regulate the expression of MyoD1 in Guanling cattle. At the same time, it is speculated that the methylation level of the MyoD1 gene promoter can affect the growth and development of Guanling cattle myoblasts, which can provide a theoretical reference for screening genetic markers to assist the improvement of Guanling cattle.

Key words: Guanling cattle, 5-Aza-2'-deoxycytidine, DNA methylation, cell cycle, apoptosis

CLC Number:

LI Xiong, TIAN Niannian, SONG Linjin, CHEN Chen, XU Houqiang. Effects of 5-Aza-dC on MyoD1 Promoter Methylation and mRNA Expression in Bovine Myoblasts[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(9): 2439-2451.

References 39

[1]	ZHAO J G, LU Z G, WANG L, et al.Plant responses to heat stress:physiology, transcription, noncoding RNA, and epigenetics[J].Int J Mol Sci, 2020, 22(1):117.
[2]	SENGUPTA A, ROY S S, CHOWDHURY S.Non-duplex G-quadruplex DNA structure:a developing story from predicted sequences to DNA structure-dependent epigenetics and beyond[J].Acc Chem Res, 2021, 54(1):46-56.
[3]	MADHANI H D.Unbelievable but true:epigenetics and chromatin in fungi[J].Trends Genet, 2021, 37(1):12-20.
[4]	HARRISON R E S, WENG K G, WANG Y X, et al.Phase separation and histone epigenetics in genome regulation[J].Curr Opin Solid State Mater Sci, 2021, 25(1):100892.
[5]	ZHANG B, BAN D M, GOU X, et al.Genome-wide DNA methylation profiles in Tibetan and Yorkshire pigs under high-altitude hypoxia[J].J Anim Sci Biotechnol, 2019, 10(1):25.
[6]	鲍正攀.调控miR-148a基因表达及5-Aza-dc处理对水牛乳腺上皮细胞增殖及泌乳相关基因表达的影响[D].南宁:广西大学, 2018.BAO Z P.Effect of regulation of miR-148a gene expression and 5-Aza-dc on the proliferation of buffalo mammary epithelial cells and their expression of genes related to laction[D].Nanning:Guangxi University, 2018.(in Chinese)
[7]	陈观娣.5-Aza-dC和放疗对宫颈癌细胞系的增殖抑制作用与P16、MGMT基因表达的关系探讨[D].广州:南方医科大学, 2018.CHEN G D.The inhibition effect of 5-Aza-dC and radiotherapy on cervical cancer cell lines proliferation and the correlation with p16 and MGMT gene expression[D].Guangzhou:Southern Medical University, 2018.(in Chinese)
[8]	GREVILLE G, LLOP E, HOWARD J, et al.5-AZA-dC induces epigenetic changes associated with modified glycosylation of secreted glycoproteins and increased EMT and migration in chemo-sensitive cancer cells[J].Clin Epigenetics, 2021, 13(1):34.
[9]	XIE M Y, YANG Y, LIU P, et al.5-aza-2'-deoxycytidine in the regulation of antioxidant enzymes in retinal endothelial cells and rat diabetic retina[J].Int J Ophthalmol, 2019, 12(1):1-7.
[10]	于翔, 王晨柳, 文逸凡, 等.DNA甲基化的表观遗传机制及其在动物遗传育种中应用[J].中国牛业科学, 2020, 46(5):59-62.YU X, WANG C L, WEN Y F, et al.Epigenetic mechanism of DNA methylation and its application in animal genetics and breeding[J].China Cattle Science, 2020, 46(5):59-62.(in Chinese)
[11]	高丽.Myostatin基因编辑牛肌肉卫星细胞成分化过程中DNA甲基化修饰的作用机制研究[D].呼和浩特:内蒙古大学, 2019.GAO L.The effect of DNA methylations on the muscle satellite cell myogenic differentiation of myostatin gene edited cattle[D].Hohhot:Inner Mongolia University, 2019.(in Chinese)
[12]	LI M Z, WU H L, LUO Z G, et al.An atlas of DNA methylomes in porcine adipose and muscle tissues[J].Nat Commun, 2012, 3:850.
[13]	李秀金, 王金辉, 黄运茂, 等.DNA甲基化影响不同猪种胚胎期肌肉发育差异的研究进展[J].中国畜牧杂志, 2020, 56(6):24-29.LI X J, WANG J H, HUANG Y M, et al.Research progresses on the role of DNA methylation in regulating distinct embryonic muscle development among pig breeds[J].Chinese Journal of Animal Science, 2020, 56(6):24-29.(in Chinese)
[14]	万火福, 韦凤英, 廖玉英, 等.鸡肌肉组织基因组DNA甲基化与肉质性状相关性分析[J].中国家禽, 2018, 40(12):5-9.WAN H F, WEI F Y, LIAO Y Y, et al.Study on the associations of genomic DNA methylation in muscle tissue with meat quality of chicken[J].China Poultry, 2018, 40(12):5-9.(in Chinese)
[15]	WEBER M, HELLMANN I, STADLER M B, et al.Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome[J].Nat Genet, 2007, 39(4):457-466.
[16]	WARDLE F C.Master control:transcriptional regulation of mammalian Myod[J].J Muscle Res Cell Motil, 2019, 40(2):211-226.
[17]	刘会敬.中外猪种胚胎不同发育时期骨骼肌转录组和全基因组甲基化分析[D].武汉:华中农业大学, 2018.LIU H J.Integration of DNA methylome and transcriptome analysis of prenatal skeletal muscles between Tongcheng and Yorkshire pigs[D].Wuhan:Huazhong Agricultural University, 2018.(in Chinese)
[18]	曹阳.杜寒杂交羊与小尾寒羊背最长肌全基因组甲基化与转录组比较分析[D].长春:吉林大学, 2017.CAO Y.Comparative analysis on genome-wide methylation and RNA-seq in longissimus dorsi muscle between dorper×small tailed Han crossbred and small tailed Han sheep[D].Changchun:Jilin University, 2017.(in Chinese)
[19]	薛倩.京海黄鸡腿肌全基因组甲基化和转录组分析研究[D].扬州:扬州大学, 2017.XUE Q.Supported by China agriculture research system (CARS-42) and priority academic program development of Jiangsu higher education institutions[D].Yangzhou:Yangzhou University, 2017.(in Chinese)
[20]	沈兰, 陈春华, 叶冬云, 等.ESR1、MYOD1和hTERT基因启动子DNA甲基化与宫颈癌放化疗敏感性的关系[J].河北医药, 2020, 42(1):33-37.SHEN L, CHEN C H, YE D Y, et al.Relationship between DNA methylation of ESR1, MYOD1, hTERT gene promoter and sensitivity to radiotherapy and chemotherapy in patients with cervical cancer[J].Hebei Medical Journal, 2020, 42(1):33-37.(in Chinese)
[21]	BORCHIELLINI M, UMMARINO S, DI RUSCIO A.The bright and dark side of DNA methylation:a matter of balance[J].Cells, 2019, 8(10):1243.
[22]	刘胜辉.黑色素瘤相关抗原MAGE-A家族在喉鳞状细胞癌中的表达及临床意义[D].石家庄:河北医科大学, 2016.LIU S H.Expressions and significance of melanoma antigen-A family in laryngeal squamous cell carcinoma[D].Shijiazhuang:Hebei Medical University, 2017.(in Chinese)
[23]	VENTÓS-ALFONSO A, YLLA G, MONTAÑES J C, et al.DNMT1 promotes genome methylation and early embryo development in cockroaches[J].iScience, 2020, 23(12):101778.
[24]	IGUCHI E, TAKAI A, TAKEDA H, et al.DNA methyltransferase 3B plays a protective role against hepatocarcinogenesis caused by chronic inflammation via maintaining mitochondrial homeostasis[J].Sci Rep, 2020, 10(1):21268.
[25]	LONG Y, UBYCH K, JAGU E, et al.FRET-based method for direct, real-time measurement of DNA methyltransferase activity[J].Bioconjug Chem, 2020, 32(1):192-198.
[26]	D'ARCY M S.Cell death:a review of the major forms of apoptosis, necrosis and autophagy[J].Cell Biol Int, 2019, 43(6):582-592.
[27]	MAJTNEROVÁ P, ROUŠAR T.An overview of apoptosis assays detecting DNA fragmentation[J].Mol Biol Rep, 2018, 45(5):1469-1478.
[28]	KIIANITSA K, ZHANG Y B, MAIZELS N.Treatment of human cells with 5-aza-dC induces formation of PARP1-DNA covalent adducts at genomic regions targeted by DNMT1[J].DNA Repair (Amst), 2020, 96:102977.
[29]	SHIRE A, LOMBERK G, LAI J P, et al.Restoration of epigenetically silenced sulfatase 1 expression by 5-Aza-2'-deoxycytidine sensitizes hepatocellular carcinoma cells to chemotherapy-induced apoptosis[J].Med Epigenet, 2015, 3(1):1-18.
[30]	李瑾.5-氮杂-2-脱氧胞苷对牛胎儿成纤维细胞和人肺癌细胞A549的影响及作用机理的研究[D].呼和浩特:内蒙古大学, 2015.LI J.The effect of 5-Aza-dC-2'-deoxycytidine on bovine fetal fibroblasts and human lung cancer A549 associated mechanism[D].Hohhot:Inner Mongolia University, 2015.(in Chinese)
[31]	吴元, 张文涛, 马文超, 等.SOX7启动子甲基化对膀胱癌增殖及凋亡的影响[J].医学研究生学报, 2019, 32(12):1285-1290.WU Y, ZHANG W T, MA W C, et al.Effect of SOX7 promoter methylation on proliferation and apoptosis of bladder cancer[J].Journal of Medical Postgraduates, 2019, 32(12):1285-1290.(in Chinese)
[32]	KIIANITSA K, ZHANG Y B, MAIZELS N.Treatment of human cells with 5-aza-dC induces formation of PARP1-DNA covalent adducts at genomic regions targeted by DNMT1[J].DNA Repair (Amst), 2020, 96:102977.
[33]	李凤珍.5-氮-2-脱氧胞苷对牛成纤维细胞周期、染色体和凋亡的影响[D].武汉:华中农业大学, 2009.LI F Z.5-Aza-2-deoxyctidine on cell cycle, chromosome and apoptosis of fibroblast[D].Wuhan:Huazhong Agricultural University, 2009.(in Chinese)
[34]	COLELLA F, SCILLITANI G, PIERRI C L.Sweet as honey, bitter as bile:mitochondriotoxic peptides and other therapeutic proteins isolated from animal tissues, for dealing with mitochondrial apoptosis[J].Toxicology, 2021, 447:152612.
[35]	WHITE T L, DESHPANDE N, KUMAR V, et al.Cell cycle re-entry and arrest in G2/M phase induces senescence and fibrosis in Fuchs Endothelial Corneal Dystrophy[J].Free Radic Biol Med, 2021, 164:34-43.
[36]	刘玉思.神经管畸形动物模型中凋亡调控因子(miR-322、PCSK9)的表达规律及作用机制的研究[D].沈阳:中国医科大学, 2020.LIU Y S.The study on the expression and mechanism of apoptosis regulatory factors (miR-322, PCSK9) in animal model of neural tube defect[D].Shenyang:China Medical University, 2020.(in Chinese)
[37]	刘丹.Rno_circ_0005139/miR-324-3p/Wnt5a调控大鼠肛门直肠畸形胚胎发育过程中细胞增殖、凋亡及作用机制的研究[D].沈阳:中国医科大学, 2020.LIU D.Rno_circ_0005139/miR-324-3p/Wnt5a regulates cell proliferation, apoptosis and the mechanism in the development of rat anorectal malformations[D].Shenyang:China Medical University, 2020.(in Chinese)
[38]	DUMONT N A, RUDNICKI M A.Characterizing satellite cells and myogenic progenitors during skeletal muscle regeneration[J].Methods Mol Biol, 2017, 1560:179-188.
[39]	MEGIORNI F, CIALFI S, MCDOWELL H P, et al.Deep Sequencing the microRNA profile in rhabdomyosarcoma reveals down-regulation of miR-378 family members[J].BMC Cancer, 2014, 14:880.

Effects of 5-Aza-dC on MyoD1 Promoter Methylation and mRNA Expression in Bovine Myoblasts

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