Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (21): 4685-4693.doi: 10.3864/j.issn.0578-1752.2021.21.017

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Interference in TP53INP2 Gene Inhibits the Differentiation of Bovine Myoblasts

DU JiaWei1(),DU XinZe1,YANG XinRan1,SONG GuiBing1,ZHAO Hui1,ZAN LinSen1,2,WANG HongBao1,2,*()   

  1. 1College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi;
    2National Beef Cattle Improvement Center, Yangling 712100, Shaanxi
  • Received:2020-09-25 Accepted:2021-03-12 Online:2021-11-01 Published:2021-11-09
  • Contact: HongBao WANG E-mail:614502306@qq.com;wanghongbao@nwsuaf.edu.cn

RichHTML

30

PDF

195

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

Table 1

Real-time PCR primer sequence"

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-R GTTATGAGGCGGAGTGTC
GAPDH NM_001034034.2 GAPDH -F AGTTCAACGGCACAGTCAAGG 124
GAPDH -R ACCACATACTCAGCACCAGCA
MYOD NM_001040478.2 MYOD-F AACCCCAACCCGATTTACC 196
MYOD-R CACAACAGTTCCTTCGCCTCT
MYOG NM_001111325.1 MYOG-F GGCGTGTAAGGTGTGTAAG 85
MYOG-R CTTCTTGAGTCTGCGCTTCT
MYH3 NM_001101835.1 MYH3-F TGAACGCCCTCTCCAAATCC 101
MYH3-R AATGAAGTGCTGTCTCGGCA

Fig. 1

The expression of TP53INP2 gene in different tissues of adult Qinchuan cattle Different lowercase letters indicate significant differences（P<0.05), different uppercase letters indicate extremely significant differences（P<0.01), and the same letters indicate that the difference is not significant"

Fig. 2

Expression of TP53INP2 in muscle cells on different days D0: day 0 of induced differentiation of myoblasts; D2: day 2 of induced differentiation of myoblasts; D4: day 4 of induced differentiation of myoblasts; D6: day 6 of induced differentiation of myoblasts; D8: induction of myoblasts Day 8 of differentiation; D10: Day 10 of differentiation of myoblasts"

Fig. 3

Detection of TP53INP2 interference efficiency **represents extremely significant differences. The same below"

Fig. 4

The phenotype of the interference group and the control group on the fourth day of induced differentiation of myoblasts The red arrow indicates the myotube formed by the fusion of muscle cells"

Fig. 5

Detection of mRNA levels of myoblast differentiation marker genes"

Fig. 6

Western Blot detection of myoblast differentiation marker genes"

Fig. 7

Relative expression of different marker proteins"

[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
[1] YANG XinRan,MA XinHao,DU JiaWei,ZAN LinSen. Expression Pattern of m6A Methylase-Related Genes in Bovine Skeletal Muscle Myogenesis [J]. Scientia Agricultura Sinica, 2023, 56(1): 165-178.
[2] SHU JingTing,SHAN YanJu,JI GaiGe,ZHANG Ming,TU YunJie,LIU YiFan,JU XiaoJun,SHENG ZhongWei,TANG YanFei,LI Hua,ZOU JianMin. Relationship Between Expression Levels of Guangxi Partridge Chicken m6A Methyltransferase Genes, Myofiber Types and Myogenic Differentiation [J]. Scientia Agricultura Sinica, 2022, 55(3): 589-601.
[3] CHEN Yu,ZHU HaoZhe,CHEN YiChun,LIU Zheng,DING Xi,GUO Yun,DING ShiJie,ZHOU GuangHong. Differentiation of Porcine Muscle Stem Cells in Three-Dimensional Hydrogels [J]. Scientia Agricultura Sinica, 2022, 55(22): 4500-4512.
[4] LIU Xin,ZHANG YaHong,YUAN Miao,DANG ShiZhuo,ZHOU Juan. Transcriptome Analysis During Flower Bud Differentiation of Red Globe Grape [J]. Scientia Agricultura Sinica, 2022, 55(20): 4020-4035.
[5] NIE XingHua, ZHENG RuiJie, ZHAO YongLian, CAO QingQin, QIN Ling, XING Yu. Genetic Diversity Evaluation of Castanea in China Based on Fluorescently Labeled SSR [J]. Scientia Agricultura Sinica, 2021, 54(8): 1739-1750.
[6] HU RongRong,DING ShiJie,GUO Yun,ZHU HaoZhe,CHEN YiChun,LIU Zheng,DING Xi,TANG ChangBo,ZHOU GuangHong. Effects of Trolox on Proliferation and Differentiation of Pig Muscle Stem Cells [J]. Scientia Agricultura Sinica, 2021, 54(24): 5290-5301.
[7] CHEN Yuan,CAI He,LI Li,WANG LinJie,ZHONG Tao,ZHANG HongPing. Alternative Splicing of TNNT3 and Its Effect on the Differentiation of MuSCs in Goat [J]. Scientia Agricultura Sinica, 2021, 54(20): 4466-4477.
[8] DU Qing,CHEN Ping,LIU ShanShan,LUO Kai,ZHENG BenChuan,YANG Huan,HE Shun,YANG WenYu,YONG TaiWen. Effect of Field Microclimate on the Difference of Soybean Flower Morphology Under Maize-Soybean Relay Strip Intercropping System [J]. Scientia Agricultura Sinica, 2021, 54(13): 2746-2758.
[9] SHI GuoLiang,WU Qiang,YANG NianWan,HUANG Cong,LIU WanXue,QIAN WanQiang,WAN FangHao. Gene Cloning, Expression Pattern and Molecular Characterization of Chitin Deacetylase 2 in Cydia pomonella [J]. Scientia Agricultura Sinica, 2021, 54(10): 2105-2117.
[10] ZHAO JiYu,REN BaiZhao,ZHAO Bin,LIU Peng,ZHANG JiWang. Relationship Between Growth and Development Characteristics and Yield Formation of Summer Maize Varieties Differing in Maturities [J]. Scientia Agricultura Sinica, 2021, 54(1): 46-57.
[11] QIN BenYuan,YANG Yang,ZHANG YanWei,LIU Min,ZHANG WanFeng,WANG HaiZhen,WU YiQi,ZHANG XueLian,CAI ChunBo,GAO PengFei,GUO XiaoHong,LI BuGao,CAO GuoQing. Isolation, Culture, Identification and Biological Characteristics of Pig Skeletal Muscle Satellite Cells [J]. Scientia Agricultura Sinica, 2020, 53(8): 1664-1676.
[12] LAI YuTing,ZHU FeiFei,WANG YiMin,GUO Hong,ZHANG LinLin,LI Xin,GUO YiWen,DING XiangBin. Effects of PSMB5 on the Proliferation and Myogenic Differentiation of Skeletal Muscle Satellite Cells [J]. Scientia Agricultura Sinica, 2020, 53(20): 4287-4296.
[13] YANG YunFei,XIN XiaoPing,LI JianDong. A Discussion on the Diffusion Pathway of Leymus Chinensis in the Natural Grassland of China Based on Differentiation in the Phenotypes and Genotypes [J]. Scientia Agricultura Sinica, 2020, 53(13): 2541-2549.
[14] GUO HongFang,NING Yue,CHENG Gong,ZAN LinSen. The Effect of Krüppel-Like Factor 3 (KLF3) Gene on Bovine Fat Deposition [J]. Scientia Agricultura Sinica, 2019, 52(7): 1272-1281.
[15] ZHU JiangJiang,LIN YaQiu,WANG Yong,LIN Sen. Expression Profile and Correlations of Kruppel Like Factors During Caprine (Capra Hircus) Preadipocyte Differentiation [J]. Scientia Agricultura Sinica, 2019, 52(13): 2341-2351.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd