Beef Cattle miR-133a Promotes the Differentiation of Myoblasts by Inhibiting PAX7

doi:10.7668/hbnxb.20192231

Abstract

Abstract: The aims to explore the molecular mechanism of bta-miR-133a involved in the regulation of longissimus dorsi development in beef cattle. The longissimus dorsi muscle of Black cattle and Luxi cattle was collected for sRNA database construction, and the data was used to screen bta-miR-133a related to the development of beef cattle skeletal muscle. Use bioinformatics analysis software to identify its conservation and predict its target genes, perform GO enrichment analysis and KEGG pathway enrichment analysis for its target genes, and verify the predicted potential target genes through the dual luciferase report test. C2C12 cells were selected for functional verification, bta-miR-133a was overexpressed and knockouted. After 48 h, 2% horse serum was used to observe the myotube differentiation process, CCK-8 to detect cell proliferation rate, Real-time quantitative polymerase chain reaction to detect the expression of cell proliferation and differentiation marker genes PCNA, CCND1, Myh1, Myod1 and target genes. The results showed that the mature sequence of bta-miR-133a was highly conserved among various species. The target gene was predicted to be PAX7. The dual luciferase report test verifies that bta-miR-133a had a target relationship with PAX7.The result of GO enrichment and KEGG pathways analysis showed that the predicted target genes were significantly enriched in the regulation of actin cytoskeleton organization tissue, the negative regulation of the classical Wnt signaling pathway, actin filament binding and other GO components, and MAPK, Ras, FoxO and other signaling pathways related to muscle development in. Overexpression of bta-miR-133a promoted myotube differentiation(P <0.01), at 72 h, reduced cell proliferation rate(P <0.01), inhibited the expression of target gene PAX7 (P <0.01);knocked out bta-miR-133a inhibited myotube differentiation(P <0.01), increased cell proliferation rate at 72 h(P <0.05), and promoted the expression of target gene PAX7 (P <0.01). In terms of cell proliferation, overexpression of bta-miR-133a reduced the expression level of its marker genes PCNA and CCND1 (P <0.05), while the knockout group was the opposite. In terms of cell differentiation, overexpression of bta-miR-133a increased the expression level of the marker genes Myh1 and Myod1 (P <0.01), while the knockout group was the opposite. In summary, bta-miR-133a may participate in the development of longissimus dorsi by targeting and negatively regulating the expression of PAX7 to inhibit the proliferation of myoblasts and promote their differentiation.

Key words: Black cattle, Luxi cattle, bta-miR-133a, PAX7, C2C12 cell

CLC Number:

S813.1

YU Kun, LIU Ruili, LIU Xianxun, BAI Xuejin, DONG Yajuan. Beef Cattle miR-133a Promotes the Differentiation of Myoblasts by Inhibiting PAX7[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(5): 226-238. doi: 10.7668/hbnxb.20192231.

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References

[1] 范永辉. miR-34b通过靶向SYISL调节成肌细胞增殖和分化[D].武汉:华中农业大学, 2019.
Fan Y H. miR-34b regulates myoblast proliferation and differentiation by targeting SYISL[D].Wuhan:Huazhong Agricultural University, 2019.
[2] Sweetman D, Goljanek K, Rathjen T, Oustanina S, Braun T, Dalmay T, Münsterberg A. Specific requirements of MRFs for the expression of muscle specific microRNAs, miR-1, miR-206 and miR-133[J]. Developmental Biology, 2008, 321(2):491-499.doi:10.1016/j.ydbio.2008.06.019.
[3] Safa A, Bahroudi Z, Shoorei H, Majidpoor J, Abak A, Taheri M, Ghafouri-Fard S. miR-1:A comprehensive review of its role in normal development and diverse disorders[J]. Biomedicine & Pharmacotherapy, 2020, 132:110903.doi:10.1016/j.biopha.2020.110903.
[4] 于太永.脂肪细胞因子leptin和TNF-α对猪骨骼肌成肌细胞增殖分化的影响及其机制[D].杨凌:西北农林科技大学, 2007.
Yu T Y. Effects of leptin and TNF-α on the porcine myoblast proliferation and differentiation and its molecular mechanism[D].Yangling:Northwest Agriculture and Forestry University, 2007.
[5] Mok G F, Lozano-Velasco E, Maniou E, Viaut C, Moxon S, Wheeler G, Münsterberg A. miR-133 mediated regulation of the hedgehog pathway orchestrates embryo myogenesis[J]. Development, 2018:145(12):159657.doi:10.1242/dev.159657.
[6] 王丽娟. MiR-1和miR-133在山羊骨骼肌组织和细胞中的表达规律及功能研究[D].合肥:安徽农业大学, 2014.
Wang L J. The expression profiles and function analysis of miR-1 and miR-133 in goat skeletal muscle tissue and cell[D].Hefei:Anhui Agricultural University, 2014.
[7] 吴宁昭. miRNA-1和miRNA-133在鸭骨骼肌发育中的表达及功能初步研究[D].扬州:扬州大学, 2017.
Wu N Z. The expression of miRNA-1 and miRNA-133 and its function on duck skeletal cell proliferation and differentiation[D].Yangzhou:Yangzhou University, 2017.
[8] 王星果, 邵芳, 龚道清, 卢祥云, 顾志良. 鸡miR-133a靶向调控BIRC5 基因的表达[J].中国农业科学, 2013, 46(7):1441-1447.doi:10.3864/j.issn.0578-1752.2013.07.015.
Wang X G, Shao F, Gong D Q, Lu X Y, Gu Z L. miR-133a targets BIRC5 to regulate its gene expression in chicken[J]. Scientia Agricultura Sinica, 2013, 46(7):1441-1447.
[9] Huang M B, Xu H, Xie S J, Zhou H, Qu L H. Insulin-like growth factor-1 receptor is regulated by microRNA-133 during skeletal myogenesis[J]. PLoS One, 2011, 6(12):e29173.doi:10.1371/journal.pone.0029173.
[10] Liu R L, Liu X X, Bai X J, Xiao C Z, Dong Y J. Different expression of lipid metabolism-related genes in Shandong black cattle and Luxi cattle based on transcriptome analysis[J]. Scientific Reports, 2020, 10:21915.doi:10.1038/s41598-020-79086-4.
[11] Liu R L, Liu X X, Bai X J, Xiao C Z, Dong Y J. Identification and characterization of circRNA in longissimus dorsi of different breeds of cattle[J]. Frontiers in Genetics, 2020, 11:565085.doi:10.3389/fgene.2020.565085.
[12] 董雅娟. 牛[M].北京:中国农业出版社, 2016:20.
Dong Y J. Cattle[M].Beijing:China Agriculture Press, 2016:20.
[13] 刘瑞莉, 吴磊, 袁玮, 柏学进, 吕娟娟, 董雅娟. MYL3 基因在肉牛肌肉生长过程中的功能研究[J].华北农学报, 2019, 34(2):221-228.doi:10.7668/hbnxb.201750982.
Liu R L, Wu L, Yuan W, Bai X J, Lü J J, Dong Y J.Function of MYL3 gene in muscle growth of cattle[J]. Acta Agriculturae Boreali-Sinica, 2019, 34(2):221-228.
[14] 吴磊, 刘瑞莉, 袁玮, 柏学进, 刘贤勋, 吕娟娟, 肖超柱, 董雅娟. CRTC2 基因在不同品种和月龄肉牛的表达分析[J].畜牧与兽医, 2019, 51(10):9-14.
Wu L, Liu R L, Yuan W, Bai X J, Liu X X, Lü J J, Xiao C Z, Dong Y J. Analysis of CRTC2 gene expression in cattle of different varieties and months of age[J]. Animal Husbandry & Veterinary Medicine, 2019, 51(10):9-14.
[15] 刘瑞莉, 吴磊, 袁玮, 柏学进, 吕娟娟, 董雅娟. 基于转录组筛选肉牛骨骼肌差异基因[J].华北农学报, 2018, 33(S1):64-72.doi:10.7668/hbnxb.2018.S1.011.
Liu R L, Wu L, Yuan W, Bai X J, Lü J J, Dong Y J. Screening of skeletal muscle differential genes based on transcriptome[J]. Acta Agriculturae Boreali-Sinica, 2018, 33(S1):64-72.
[16] Cesana M, de Cacchiarelli D, Legnini I, Santini T, Sthandier O, Chinappi M, Tramontano A, Bozzoni I.A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA[J]. Cell, 2011, 147(2):358-369.doi:10.1016/j.cell.2011.09.028.
[17] Ma X Y, Wei D W, Cheng G, Li S J, Wang L, Wang Y N, Wang X Y, Zhang S, Wang H B, Zan L S. Bta-miR-130a/b regulates preadipocyte differentiation by targeting PPARG and CYP2U1 in beef cattle[J]. Molecular and Cellular Probes, 2018, 42:10-17.doi:10.1016/j.mcp.2018.10.002.
[18] Dey B K, Gagan J, Dutta A. miR-206 and-486 induce myoblast differentiation by downregulating Pax7[J]. Molecular and Cellular Biology, 2011, 31(1):203-214.doi:10.1128/mcb.01009-10.
[19] 孙加节. 秦川牛肌肉与脂肪组织发育相关miRNA鉴定及miR-10020调控机制解析[D].杨凌:西北农林科技大学, 2015.
Sun J J. Identification of microRNAs from Chinese Qinchuan bovine muscle and adipose tissues and functional analysis of miR-10020 during myogenesis[D].Yangling:Northwest Agriculture and Forestry University, 2015.
[20] 王艳红. 山羊不同生长发育阶段肌肉组织miRNA的鉴定及其调控机制研究[D].杨凌:西北农林科技大学, 2015.
Wang Y H. Identification of muscle-related miRNA from skeletal muscle in different development stage goat and analysis on their regulation roles[D].Yangling:Northwest Agriculture and Forestry University, 2015.
[21] 郭晓萍. 猪骨骼肌miRNA转录组分析及miR-486功能的初步研究[D].南宁:广西大学, 2015.
Guo X P. Transcriptome analysis of porcine skeletal muscle miRNAs and primary study of miR-486 function[D].Nanning:Guangxi University, 2015.
[22] 娄秋宏. gga-miR-206在金茅黑鸡胚胎骨骼肌发育中的表达分析及靶基因验证[D].扬州:扬州大学, 2019.
Lou Q H. Expression of gga-miR-206 in different stages of skeletal muscle development in Jinmao black chicken embryos and its targets identification[D].Yangzhou:Yangzhou University, 2019.
[23] Ling Y H, Sui M H, Zheng Q, Wang K Y, Wu H, Li W Y, Liu Y, Chu M X, Fang F G, Xu L N. miR-27b regulates myogenic proliferation and differentiation by targeting Pax3 in goat[J]. Scientific Reports, 2018, 8:3909.doi:10.1038/s41598-018-22262-4.
[24] Gagan J, Dey B K, Layer R, Yan Z, Dutta A. MicroRNA-378 targets the myogenic repressor MyoR during myoblast differentiation[J]. Journal of Biological Chemistry, 2011, 286(22):19431-19438.doi:10.1074/jbc.M111.219006.
[25] 化朝举. miR-378a对骨骼肌肌纤维类型及其代谢的调控机理[D].北京:中国农业科学院, 2016.
Hua C J. Molecular mechanism of miR-378a regulating myofiber type and metabolism of skeletal muscle[D]. Beijing:Chinese Academy of Agricultural Sciences, 2016.
[26] 庚跃琦, 孔令珍, 贾垂明, 李丹丹. GPM6A在肺癌组织中的表达差异及临床意义[J].现代肿瘤医学, 2019, 27(9):1546-1550.doi:10.3969/j.issn.1672-4992.2019.09.019.
Geng Y Q, Kong L Z, Jia C M, Li D D. Analysis of expression profile of GPM6A and clinical significance in lung cancer tissues[J]. Journal of Modern Oncology, 2019, 27(9):1546-1550.
[27] Wang X C, Wang C, Xi L, Yu Z Q. Rap2c as a novel biomarker for predicting poor prognosis in glioma[J]. OncoTargets and Therapy, 2020, 13:3073-3083.doi:10.2147/OTT.S247731.
[28] Wang Z X, Huang C P, Zhang A B, Lu C, Liu L F. Overexpression of circRNA_100290 promotes the progression of laryngeal squamous cell carcinoma through the miR-136-5p/RAP2C axis[J]. Biomedicine & Pharmacotherapy, 2020, 125:109874.doi:10.1016/j.biopha.2020.109874.
[29] Zhu X Y, Qiu J X, Zhang T, Yang Y P, Guo S, Li T S, Jiang K F, Zahoor A, Deng G Z, Qiu C W. MicroRNA-188-5p promotes apoptosis and inhibits cell proliferation of breast cancer cells via the MAPK signaling pathway by targeting Rap2c[J]. Journal of Cellular Physiology, 2020, 235(3):2389-2402.doi:10.1002/jcp.29144.
[30] Zhu T J, Chen Y T, Liu Z J, Leng Y X, Tian Y. Expression profiles and prognostic significance of AFTPH in different tumors[J]. FEBS Open Biochemistry, 2020, 10(12):2666-2677.doi:10.1002/2211-5463.13003.
[31] 孙多临, 冯汉卿, 赵恒, 陈胜男, 李松美, 王春生. 绵羊Pax7 基因慢病毒示踪载体的构建与表达检测[J].黑龙江畜牧兽医, 2020(19):11-15, 165.doi:10.13881/j.cnki.hljxmsy.2019.12.0060.
Sun D L, Feng H Q, Zhao H, Chen S N, Li S M, Wang C S. Construction and expression detection of Pax7 gene lentiviral vector in sheep[J]. Heilongjiang Animal Science and Veterinary Medicine, 2020(19):11-15, 165.
[32] Relaix F, Rocancourt D, Mansouri A, Buckingham M. A Pax3/Pax7-dependent population of skeletal muscle progenitor cells[J]. Nature, 2005, 435(7044):948-953.doi:10.1038/nature03594.
[33] Relaix F, Montarras D, Zaffran S, Gayraud-Morel B, Rocancourt D, Tajbakhsh S, Mansouri A, Cumano A, Buckingham M. Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells[J]. The Journal of Cell Biology, 2006, 172(1):91-102.doi:10.1083/jcb.200508044.
[34] Oustanina S, Hause G, Braun T. Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification[J]. The EMBO Journal, 2004, 23(16):3430-3439.doi:10.1038/sj.emboj.7600346.
[35] Tureckova J, Wilson E M, Cappalonga J L, Rotwein P. Insulin-like growth factor-mediated muscle differentiation:collaboration between phosphatidylinositol 3-kinase-akt-signaling pathways and myogenin[J]. Journal of Biological Chemistry, 2001, 276(42):39264-39270.doi:10.1074/jbc.M104991200.
[36] Yu M L, Wang H, Xu Y L, Yu D B, Li D F, Liu X H, Du W X. Insulin-like growth factor-1(IGF-1) promotes myoblast proliferation and skeletal muscle growth of embryonic chickens via the PI3K/Akt signalling pathway[J]. Cell Biology International, 2015, 39(8):910-922.doi:10.1002/cbin.10466.
[37] 石斌刚. 天祝白牦牛肌肉生长和肌内脂肪沉积相关基因筛选与鉴定[D].兰州:甘肃农业大学, 2020.
Shi B G. Identification of genes associated with muscle growth and intramuscular fat deposition in Tianzhu white yak[D].Lanzhou:Gansu Agricultural University, 2020.
[38] 张维娅. 不同年龄骨骼肌卫星细胞的分化能力及调控机制研究[D].武汉:华中农业大学, 2018.
Zhang W Y. The differentiation and regulation mechanism of skeletal muscle satellite cells at different ages[D].Wuhan:Huazhong Agricultural University, 2018.
[39] von Maltzahn J, Jones A E, Parks R J, Rudnicki M A. Pax7 is critical for the normal function of satellite cells in adult skeletal muscle[J]. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(41):16474-16479.doi:10.1073/pnas.1307680110.
[40] Günther S, Kim J, Kostin S, Lepper C, Fan C M, Braun T. Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells[J]. Cell Stem Cell, 2013, 13(5):590-601.doi:10.1016/j.stem.2013.07.016.
[41] Buckingham M, Relaix F. PAX3 and PAX7 as upstream regulators of myogenesis[J]. Seminars in Cell & Developmental Biology, 2015, 44:115-125.doi:10.1016/j.semcdb.2015.09.017.
[42] Kong D L, He M, Yang L, Zhou R T, Yan Y Q, Liang Y, Teng C B. MiR-17 and miR-19 cooperatively promote skeletal muscle cell differentiation[J]. Cellular and Molecular Life Sciences, 2019, 76(24):5041-5054.doi:10.1007/s00018-019-03165-7.
[43] 孔德麟. miR-17-92家族对骨骼肌成肌细胞分化调节机制的研究[D].哈尔滨:东北林业大学, 2019.
Kong D L. Mechanism study of MiR-17-92 in regulating skeletal muscle cell differentiation[D].Harbin:Northeast Forestry University, 2019.
[44] 张蔚然. miR-143对牛骨骼肌卫星细胞成肌分化的调控作用研究[D].天津:天津农学院, 2017.
Zhang W R. Study on the regulation mechanism of microRNA-143 in the proliferation and myogenic differentiation process of bovine skeletal muscle satellite cells[D].Tianjin:Tianjin Agricultural University, 2017.
[45] 代阳. microRNA-128对牛骨骼肌卫星细胞增殖和成肌分化的调控机制研究[D].天津:天津农学院, 2016.
Dai Y. Study on the regulation mechanism of microRNA-128 in the proliferation and myogenic differentiation process of bovine skeletal muscle satellite cells[D].Tianjin:Tianjin Agricultural University, 2016.
[46] 邬明丽. miR-24-3p调控肌细胞增殖与凋亡的机制研究[D].杨凌:西北农林科技大学, 2019.
Wu M L. Regulating mechanism of miR-24-3p in myoblasts proliferation and apoptosis[D].Yangling:Northwest Agriculture and Forestry University, 2019.
[47] 邢义珅. NCAGP 基因对牛胎儿骨骼肌来源的成肌细胞有丝分裂和成肌分化的影响及机理研究[D].北京:中国农业科学院, 2018.
Xing Y S. The effects of NCAPG on the mitosis and myogenic differentiation of myoblasts derived from fetal bovine skeletal muscle and mechanism study[D].Beijing:Chinese Academy of Agricultural Sciences, 2018.
[48] 秦瑞峰, 顾晓明, 秦晓春. 骨骼肌肌细胞增殖及分化过程中凋亡现象[J].第四军医大学学报, 2001, 22(1):20-22.doi:10.3321/j.issn:1000-2790.2001.01.006.
Qin R F, Gu X M, Qin X C. Apoptosis in proliferation and differentiation of skeletal muscle cells[J]. Journal of the Fourth Military Medical University, 2001, 22(1):20-22.
[49] 陈永乐, 周光前, 邓宇斌, 智伟, 谢彗琪, 邓力, 杨志明, 王亚柱. C2C12成肌细胞体外诱导分化为肌管的实验[J].中山大学学报(医学科学版), 2008, 29(1):10-15.doi:10.3321/j.issn:1672-3554.2008.01.003.
Chen Y L, Zhou G Q, Deng Y B, Zhi W, Xie H Q, Deng L, Yang Z M, Wang Y Z. In vitro inducement of C2C12 myoblasts into differentiation of myotubes[J]. Journal of Sun Yat-Sen University (Medical Sciences), 2008, 29(1):10-15.
[50] Tian H Z, She Z W, Gao X J, Wang W P, Tian H. MicroRNA-31 regulates dental epithelial cell proliferation by targeting Satb2[J]. Biochemical and Biophysical Research Communications, 2020, 532(2):321-328.doi:10.1016/j.bbrc.2020.07.138.
[51] 王萌. miR-34c通过YY1调控骨骼肌细胞增殖及分化的机制研究[D].北京:中国农业大学, 2017.
Wang M. miR-34c regulates the proliferation and differentiation of skeletal muscle cells by targeting YY1[D].Beijing:China Agricultural University, 2017.
[52] 韩福慧, 李倩, 栾兆进, 王国义, 柳楠, 贺建宁, 薛明. miR-374b及其靶基因Myf6 调控成肌细胞增殖分化的研究[J].中国畜牧杂志, 2020, 56(8):95-100.doi:10.19556/j.0258-7033.20190726-07.
Han F H, Li Q, Luan Z J, Wang G Y, Liu N, He J N, Xue M.Proliferation and differentiation regulation of miR-374b on ovine myoblasts by targeting Myf6 gene[J]. Chinese Journal of Animal Science, 2020, 56(8):95-100.
[53] 侯欣华. 猪骨骼肌相关miRNA的鉴定及miR-378对肌肉发育调控的研究[D].南京:南京农业大学, 2011.
Hou X H. Identification of porcine skeletal muscle related miRNA and regulation role of miR-378 in myogenesis[D].Nanjing:Nanjing Agricultural University, 2011.
[54] 刘建华, 梁煜, 车雨彤, 王凤, 刘旭莹, 赵弼时, 王强, 安丽霞, 乔利英, 刘文忠. miR-128-1-sp对绵羊前体脂肪细胞增殖和分化调节作用的研究[J].畜牧兽医学报, 2020, 51(6):1207-1218.doi:10.11843/j.issn.0366-6964.2020.06.005.
Liu J H, Liang Y, Che Y T, Wang F, Liu X Y, Zhao B S, Wang Q, An L X, Qiao L Y, Liu W Z.Study on the regulation of miR-128-1-sp in proliferation and differentiation of ovine preadipocyte[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6):1207-1218.
[55] 宋成创. IGF2 基因来源的IGF2 AS和miR-483调控牛骨骼肌细胞增殖分化机制研究[D].杨凌:西北农林科技大学, 2019.
Song C C. IGF2 gene-derived IGF2 AS and miR-483 regulate proliferation and differentiation of bovine skeletal muscle cells[D].Yangling:Northwest Agriculture and Forestry University, 2019.
[56] Nguyen H T, Frasch M. MicroRNAs in muscle differentiation:Lessons from Drosophila and beyond[J]. Current Opinion in Genetics & Development, 2006, 16(5):533-539.doi:10.1016/j.gde.2006.08.010.
[57] Chen J F, Mandel E M, Thomson J M, Wu Q L, Callis T E, Hammond S M, Conlon F L, Wang D Z.The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation[J]. Nature Genetics, 2006, 38(2):228-233.doi:10.1038/ng1725.
[58] Boutz P L, Chawla G, Stoilov P, Black D L. MicroRNAs regulate the expression of the alternative splicing factor nPTB during muscle development[J]. Genes & Development, 2007, 21(1):71-84.doi:10.1101/gad.1500707.
[59] Chen X, Wang K H, Chen J N, Guo J G, Yin Y, Cai X, Guo X, Wang G Q, Yang R, Zhu L Y, Zhang Y, Wang J, Xiang Y, Weng C Y, Zen K, Zhang J F, Zhang C Y. In vitro evidence suggests that miR-133a-mediated regulation of uncoupling protein 2(UCP2) is an indispensable step in myogenic differentiation[J]. The Journal of Biological Chemistry, 2009, 284(8):5362-5369.doi:10.1074/jbc.M807523200.

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