干扰TP53INP2抑制牛成肌细胞分化

doi:10.3864/j.issn.0578-1752.2021.21.017

摘要/Abstract

摘要：

【背景】肌肉可以维持哺乳动物运动功能并调节机体代谢,它的数量和分布对肉质有重要影响,骨骼肌的生长发育及其遗传特性在很大程度上影响甚至决定家畜的产肉量和肉品质,研究骨骼肌的生长发育具有重要意义。TP53INP2对自噬的调控作用以及对前体脂肪细胞分化的调控机制已有研究,而对于牛成肌细胞分化的影响尚未报道。【目的】探究TP53INP2对秦川牛成肌细胞分化的影响,为肉牛肉用性状分子育种工作提供理论依据。【方法】利用实时荧光定量PCR（real-time fluorescence quantitative PCR,RT-qPCR）技术检测了TP53INP2在24月龄秦川牛不同组织中的表达特性,同时分析了其在体外培养的牛骨骼肌成肌细胞不同分化时期的表达规律;合成TP53INP2基因siRNA并转染秦川牛成肌细胞,转染后12h进行诱导分化,观察成肌细胞表型变化,并利用RT-qPCR技术和Western Blot技术分别检测其在诱导分化第四天时分化标志基因和蛋白的表达情况。【结果】1.RT-qPCR结果显示,TP53INP2在成年秦川牛背最长肌组织中表达量最高,在小肠组织中表达量最低。TP53INP2在成年牛心脏、肝脏、肾脏、网胃、瘤胃中表达量较高,在其余组织中表达量较低（与背最长肌相比）。2.随着成肌细胞的分化,该基因在第0—4天表达量呈上升趋势且在第4天达到峰值,随后表达量呈下降趋势。3.在成肌细胞中干扰TP53INP2后,试验组肌管的数量和长度均显著低于对照组。4.干扰TP53INP2并提取细胞总RNA,RT-qPCR结果显示,成肌细胞分化标志基因肌细胞生成素（MYOG）和肌球重链蛋白3（MYH3）相对于对照组表达量显著降低。5.干扰TP53INP2并提取细胞总蛋白,Western Blot结果显示,试验组MYOG、MYH3、MYOD的蛋白表达量均降低且与对照组相比差异极显著。【结论】干扰TP53INP2对牛成肌细胞的分化具有抑制作用,提示该基因可能对秦川牛肌肉组织的生长发育具有重要的调控作用,可对其进行深入的功能研究以期用于肉牛的分子育种实践中。

关键词: TP53INP2, 分化, 成肌细胞, 秦川牛

Abstract:

【Background】Muscle can maintain the motor function of mammals and regulate body metabolism. Its quantity and distribution have an important influence on meat quality. The growth and development of skeletal muscle and its genetic characteristics influence and even determine the meat production and meat quality to a large extent. It is of great significance to study the growth and development of skeletal muscle. The regulatory effect of TP53INP2 on autophagy and the regulation mechanism on the differentiation of preadipocytes have been studied, but whether it affects the differentiation of bovine myoblasts has not been reported. 【Objective】 This study aims to explore the effect of TP53INP2 on the differentiation of Qinchuan bovine myoblasts in order to provide a theoretical basis for the molecular breeding of beef cattle meat traits. 【Method】 The real-time fluorescence quantitative PCR (RT-qPCR) technology was used to detect the expression characteristics of TP53INP2 in different tissues of Qinchuan cattle at 24 months of age. At the same time, the expression patterns of bovine skeletal myoblasts cultured in vitro at different stages of differentiation were analyzed. TP53INP2 gene siRNA was synthesized and transfected to Qinchuan bovine myoblasts, cells were induced to differentiation 12 hours after transfection, phenotypic changes of myoblasts were observed at different time pionts, and RT-qPCR and Western Blot technologies were performed respectively to detect the expression of differentiation marker genes and proteins on the fourth day of induced differentiation. 【Result】 1. RT-qPCR results showed that the expression level of TP53INP2 was the highest in adult Qinchuan cattle Longissimus dorsi muscle tissue and the lowest in the small intestine tissue. It is higher in adult bovine heart, liver, kidney, reticulum and rumen, and lower in other tissues (compared with longissimus dorsi). 2. With the differentiation of myoblasts, the expression of this gene increased from 0 to 4 days and reached a peak on the 4th day, and then decreased. 3. After interfering with TP53INP2 siRNA in myoblasts, the number and length of myotubes in the test group were significantly lower than those in the control group. 4. RT-qPCR results showed that the expression of myoblast differentiation marker genes myogenin (MYOG) and myosin heavy chain protein 3 (MYH3) was significantly lower than that of the control group. Western Blot results showed that the protein expressions of MYOG, MYH3 and MYOD in the test group were reduced and the differences were extremely significant compared with the control group.【Conclusion】Interfering of TP53INP2 has an inhibitory effect on the differentiation of bovine myoblasts, suggesting that this gene may have an important regulatory effect on the growth and development of Qinchuan cattle muscle tissue, and it can be used for in-depth functional research for molecular breeding of beef cattle practice.

Key words: TP53INP2, differentiation, myoblasts, Qinchuan cattle

杜嘉伟,杜鑫泽,杨昕冉,宋贵兵,赵慧,昝林森,王洪宝. 干扰TP53INP2抑制牛成肌细胞分化[J]. 中国农业科学, 2021, 54(21): 4685-4693.

DU JiaWei,DU XinZe,YANG XinRan,SONG GuiBing,ZHAO Hui,ZAN LinSen,WANG HongBao. Interference in TP53INP2 Gene Inhibits the Differentiation of Bovine Myoblasts[J]. Scientia Agricultura Sinica, 2021, 54(21): 4685-4693.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.21.017

https://www.chinaagrisci.com/CN/Y2021/V54/I21/4685

图/表 8

表1

图1

图2

图3

图4

图5

图6

图7

参考文献 30

[1]	SALA D, ZORZANO A. Is TP53INP2 a critical regulator of muscle mass. Current opinion in clinical nutrition and metabolic care, 2015, 18(3):234-239. doi: 10.1097/MCO.0000000000000163. doi: 10.1097/MCO.0000000000000163
[2]	陈俐静, 陈卓, 李娜, 孙亚伟, 李红波, 宋雯雯, 张杨, 姚刚. 新疆褐牛与安格斯牛胴体及肉质性状及脂代谢相关基因表达差异比较. 中国农业科学, 2020(22):4700-4709.
	CHEN L J, CHEN Z, LI N, SUN Y W, LI H B, SONG W W, ZHANG Y, YAO G. Comparison of the carcass and beef quality traits with the expression of the lipid metabolism related genes between Xinjiang brown cattle and Angus beef cattle. Scientia Agricultura Sinica, 2020(22):4700-4709. (in Chinese)
[3]	宁越, 米雪, 陈星伊, 邵建航, 昝林森. SMAD1基因的沉默和过表达及对秦川牛原代成肌细胞生肌的影响. 中国农业科学, 2019, 52(10):1818-1829. doi: 10.3864/j.issn.0578-1752.2019.10.014. doi: 10.3864/j.issn.0578-1752.2019.10.014
	NING Y, MI X, CHEN X Y, SHAO J H, ZAN L S. Silencing and overexpressing SMAD family member 1(SMAD1) gene and its effect on myogenesis in primary myoblast of Qinchuan cattle(Bos taurus). Scientia Agricultura Sinica, 2019, 52(10):1818-1829. doi: 10.3864/j.issn.0578-1752.2019.10.014. (in Chinese) doi: 10.3864/j.issn.0578-1752.2019.10.014
[4]	吴垚群, 陈少康, 盛熙晖, 齐晓龙, 王相国, 倪和民, 郭勇, 王楚端, 邢凯. 用高通量测序技术研究松辽黑猪与长白猪背最长肌mRNA和lncRNA的差异表达. 中国农业科学, 2020(4):836-847.
	WU Y Q, CHEN S K, SHENG X H, QI X L, WANG X G, NI H M, GUO Y, WANG C D, XING K. Differential expression of mRNA and lncRNA in longissimus dorsi muscle of Songliao black pig and Landrace pig based on high-throughput sequencing technique. Scientia Agricultura Sinica, 2020(4):836-847. (in Chinese)
[5]	SUN Y J, LIU K P, HUANG Y Z, LAN X Y, CHEN H. Differential expression of FOXO1 during development and myoblast differentiation of Qinchuan cattle and its association analysis with growth traits. Science China Life Sciences, 2018, 61(7):826-835. doi: 10.1007/s11427-017-9205-1. doi: 10.1007/s11427-017-9205-1
[6]	HE M Z, ZHAO Y L, YI H Q, SUN H, LIU X D, MA S M. The combination of TP53INP1, TP53INP₂ and AXIN2: potential biomarkers in papillary thyroid carcinoma. Endocrine, 2015, 48(2):712-717. doi: 10.1007/s12020-014-0341-8. doi: 10.1007/s12020-014-0341-8
[7]	OKAMURA S, ARAKAWA H, TANAKA T, NAKANISHI H, NG C C, TAYA Y, MONDEN M, NAKAMURA Y. p53DINP1, a p53-inducible gene, regulates p53-dependent apoptosis. Molecular Cell, 2001, 8(1):85-94. doi: 10.1016/s1097-2765(01)00284-2. doi: 10.1016/s1097-2765(01)00284-2
[8]	ZHANG W Y, LI P W, WANG S J, GONG C, W LI, M XUE, SU X T, WANG Y N, ZAN L S. TP53INP2 promotes bovine adipocytes differentiation through autophagy activation. Animals, 2019, 9(12):1060. doi: 10.3390/ani9121060. doi: 10.3390/ani9121060
[9]	HUANG R, LIU W. Identifying an essential role of nuclear LC3 for autophagy. Autophagy, 2015, 11(5):852-853. doi: 10.1080/15548627.2015.1038016. doi: 10.1080/15548627.2015.1038016
[10]	XU Y F, WAN W, SHOU X, HUANG R, YOU Z Y, SHOU Y H, WANG L L, ZHOU T H, LIU W. TP53INP2/DOR, a mediator of cell autophagy, promotes rDNA transcription via facilitating the assembly of the POLR1/RNA polymerase I preinitiation complex at rDNA promoters. Autophagy, 2016, 12(7):1118-1128. doi: 10.1080/15548627.2016.1175693. doi: 10.1080/15548627.2016.1175693
[11]	SANCHO A, DURAN J, GARCÍA-ESPAÑA A, C MAUVEZIN, ALEMU E A, LAMARK T, MACIAS M J, DESALLE R, ROYO M, SALA D. DOR/Tp53inp2 and Tp53inp1 constitute a Metazoan gene family encoding dual regulators of autophagy and transcription. PLoS ONE, 2012. doi: 10.1371/journal.pone.0034034. doi: 10.1371/journal.pone.0034034
[12]	DAVID S, SAŠKA I, NATÀLIA P, VICENT R, JORDI D, DANIEL B, SAADET T, MARTINE L, HUBERT V, MONIKA K K. Autophagy- regulating TP53INP2 mediates muscle wasting and is repressed in diabetes. The Journal of Clinical Investigation, 2014, 124(5):1914-1927. doi: 10.1172/JCI72327. doi: 10.1172/JCI72327
[13]	LEE Y K, JUN Y W, CHOI H E, HUH Y H, KAANG B K, JANG D J, LEE J A. Development of LC3/GABARAP sensors containing a LIR and a hydrophobic domain to monitor autophagy. EMBO Journal, 2017, 36(8):1100-1116. doi: 10.15252/embj.201696315. doi: 10.15252/embj.201696315
[14]	NOWAK J, IOVANNA J L. TP53INP₂ is the new guest at the table of self-eating. Autophagy, 2009, 5(3):383-384. doi: 10.4161/auto.5.3.7698. doi: 10.4161/auto.5.3.7698
[15]	JONATHAN N, CENDRINE A, JOËL T L, PIERRE P, MARIE- JOSÈPHE P, INÉS V M, GUILLERMO V, JEAN-CHARLES D, LUCIO I J. The TP53INP2 protein is required for autophagy in mammalian cells. Molecular Biology of the Cell, 2009, 20(3):870-881. doi: 10.1091/mbc.e08-07-0671. doi: 10.1091/mbc.e08-07-0671
[16]	GOLDMAN S, ZHANG Y, JIN S. Autophagy and adipogenesis: implications in obesity and type II diabetes. Autophagy, 2010, 6(1):179-181. doi: 10.4161/auto.6.1.10814. doi: 10.4161/auto.6.1.10814
[17]	KOVSAN J, BLÜHER M, TARNOVSCKI T, KLÖTING N, KIRSHTEIN B, MADAR L, SHAI I, GOLAN R, HARMAN- BOEHM I, SCHÖN M R, GREENBERG A S, ELAZAR Z, BASHAN N, RUDICH A. Altered autophagy in human adipose tissues in obesity. The Journal of Clinical Endocrinology and Metabolism, 2011, 96(2):E268-E277. doi: 10.1210/jc.2010-1681. doi: 10.1210/jc.2010-1681
[18]	FROMM-DORNIEDEN C, LYTOVCHENKO O, VON DER HEYDE S, BEHNKE N, HOGL S, BERGHOFF J, KÖPPER F, OPITZ L, RENNE U, HOEFLICH A, BEISSBARTH T, BRENIG B, BAUMGARTNER B G. Extrinsic and intrinsic regulation of DOR/TP53INP₂ expression in mice: effects of dietary fat content, tissue type and sex in adipose and muscle tissues. Nutrition & Metabolism, 2012, 9(1):86. doi: 10.1186/1743-7075-9-86. doi: 10.1186/1743-7075-9-86
[19]	HU Y, LI X, XUE W, PANG J, MENG Y, SHEN Y, XU Q. TP53INP₂-related basal autophagy is involved in the growth and malignant progression in human liposarcoma cells. Biomedicine & Pharmacotherapy, 2017, 88:562-568. doi: 10.1016/j.biopha.2017.01.110. doi: 10.1016/j.biopha.2017.01.110
[20]	赵艳芳. Rybp基因对秦川牛成肌细胞分化作用的研究[D]. 杨凌:西北农林科技大学. 2017.
	ZHAO Y F. The effect of Rybp gene on the differentiation of Qinchuan bovine myoblasts[D]. Yangling: Northwest A & F university. 2017. (in Chinese)
[21]	赵艳芳, 张乐, 王亚宁, 宁越, 王洪宝, 昝林森. 牛Rybp基因的表达特性分析. 西北农林科技大学学报(自然科学版), 2018, 46(5):8-15, 29. doi: 10.13207/j.cnki.jnwafu.2018.05.002. doi: 10.13207/j.cnki.jnwafu.2018.05.002
	ZHAO Y F, ZHANG L, WANG Y N, NING Y, WANG H B, ZAN L S. Expression characteristics of cattle Rybp gene. Journal of Northwest A&F University(Natural Science Edition), 2018, 46(5):8-15, 29. doi: 10.13207/j.cnki.jnwafu.2018.05.002. (in Chinese) doi: 10.13207/j.cnki.jnwafu.2018.05.002
[22]	李佩韦, 吴森, 王亚宁, 王洪宝, 昝林森. PLIN2基因在秦川肉牛皮下脂肪组织中的表达规律及基因多态性与肉质性状的关联分析. 中国畜牧兽医, 2018(6):1580-1589.
	LI P W, WU S, WANG Y N, WANG H B, ZAN L S. Expression patterns of PLIN₂ gene in subcutaneous adipose tissue and association analysis of single nucleotide polymorphisms with meat quality traits in Qinchuan cattle. China Animal Husbandry & Veterinary Medicine, 2018(6):1580-1589. (in Chinese)
[23]	马懿磊. 黄牛IGF1R基因遗传变异及其对成肌细胞增殖、分化的影响[D]. 杨凌:西北农林科技大学, 2019.
	MA Y L. Genetic variation of cattle IGF1R gene and its effect on the proliferation and differentiation of myoblasts[D]. Yangling: Northwest A & F university. 2019. (in Chinese)
[24]	HINDI S M, TAJRISHI M M, KUMAR A. Signaling mechanisms in mammalian myoblast fusion. Science Signaling, 2013, 6(272): re2. doi: 10.1126/scisignal.2003832. doi: 10.1126/scisignal.2003832
[25]	BAUMGARTNER B G, ORPINELL M, DURAN J, RIBAS V, BURGHARDT H E, BACH D, VILLAR A V, PAZ J C, GONZÁLEZ M, CAMPS M, et al. Identification of a novel modulator of thyroid hormone receptor-mediated action. PLoS ONE, 2007, 2(11). doi: 10.1371/journal.pone.0001183. doi: 10.1371/journal.pone.0001183
[26]	ZHANG Z T, XU F, ZHANG Y N, LI W, YIN Y H, ZHU C Y, DU L X, ELSAYED A K, LI B C. Cloning and expression of MyoG gene from Hu sheep and identification of its myogenic specificity. Molecular Biology Reports, 2014, 41(2):1003-1013. doi: 10.1007/s11033-013-2945-0. doi: 10.1007/s11033-013-2945-0
[27]	SCALA M, ACCOGLI A, DE GRANDIS E, ALLEGRI A, BAGOWSKI C P, SHOUKIER M, MAGHNIE M, CAPRA V. A novel pathogenic MYH₃ mutation in a child with Sheldon-Hall syndrome and vertebral fusions. American Journal of Medical Genetics Part A, 2018, 176(3):663-667. doi: 10.1002/ajmg.a.38593. doi: 10.1002/ajmg.a.38593
[28]	WARDLE F C. Master control: transcriptional regulation of mammalian Myod. Journal of Muscle Research and Cell Motility, 2019, 40(2):211-226. doi: 10.1007/s10974-019-09538-6. doi: 10.1007/s10974-019-09538-6
[29]	王欣悦, 石田培, 赵志达, 胡文萍, 尚明玉, 张莉. 基于绵羊胚胎骨骼肌蛋白质组学的PI3K-AKT信号通路分析. 中国农业科学, 2020(14):2956-2963.
	WANG X Y, SHI T P, ZHAO Z D, HU W P, SHANG M Y, ZHANG L. The analysis of PI3K-AKT signal pathway based on the proteomic results of sheep embryonic skeletal muscle. Scientia Agricultura Sinica, 2020(14):2956-2963. (in Chinese)
[30]	KIM J H, CHOI T G, PARK S, YUN H R, NGUYEN N N Y, JO Y H, JANG M, KIM J, KIM J, KANG I, HA J, MURPHY M P, TANG D G, KIM S S. Mitochondrial ROS-derived PTEN oxidation activates PI3K pathway for mTOR-induced myogenic autophagy. Cell Death and Differentiation, 2018, 25(11):1921-1937. doi: 10.1038/s41418-018-0165-9. doi: 10.1038/s41418-018-0165-9

基因名称 Gene name	GenBank号 GenBank No.	引物名称 Primer name	引物序列 (5′—3′） Primer sequence (5′--3′）	产物长度 Product length (bp)
TP53INP2	XM_003586843.5	TP53INP2-F	GCGGCTGTAGACTCAAAG	130
TP53INP2	XM_003586843.5	TP53INP2-R	GTTATGAGGCGGAGTGTC	130
GAPDH	NM_001034034.2	GAPDH -F	AGTTCAACGGCACAGTCAAGG	124
GAPDH	NM_001034034.2	GAPDH -R	ACCACATACTCAGCACCAGCA	124
MYOD	NM_001040478.2	MYOD-F	AACCCCAACCCGATTTACC	196
MYOD	NM_001040478.2	MYOD-R	CACAACAGTTCCTTCGCCTCT	196
MYOG	NM_001111325.1	MYOG-F	GGCGTGTAAGGTGTGTAAG	85
MYOG	NM_001111325.1	MYOG-R	CTTCTTGAGTCTGCGCTTCT	85
MYH3	NM_001101835.1	MYH3-F	TGAACGCCCTCTCCAAATCC	101
MYH3	NM_001101835.1	MYH3-R	AATGAAGTGCTGTCTCGGCA	101