Effects of miR-133a on the Proliferation and Differentiation of Bovine (Bos taurus) Skeletal Muscle-derived Satellite Cells
LI Yan1, YANG Xu1, CHEN Ming-Ming1, GUO Hong1, DING Xiang-Bin1, LIU Xin-Feng1,2,3,*
1 Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry/College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300384, China; 2 Key Laboratory of Education for Conservation and Utilization of Special Biological Resources in the Western China, Ningxia University, Yinchuan 750021, China; 3 School of Life Science, Ningxia University, Yinchuan 750021, China
Abstract：miR-133a is the microRNA (miRNA) specifically expressed in skeletal muscle and myocardium, but its role in the proliferation and differentiation of skeletal muscle-derived satellite cells (MDSCs) in Bos taurus is unknown. In order to further understand the effect of miR-133a on the proliferation and differentiation in bovine MDSCs, and enrich the molecular genetic mechanism affecting the growth and development of bovine muscle, in this study, based on the established in vitro differentiation model of Bos taurus MDSCs, qRT-PCR method was used to detect the temporal expression profiles of miR-133a, myogenin (MyoG), myogenic differentiation (MyoD) and myosin heavy chain (MHC) in MDSCs. Then mimic and inhibitors of miR-133a were used to transfect BMSCs to construct a model of miR-133a overexpressing or inhibiting, and transfection effects on MDSCs proliferation were detected by 5-ethynyl-2'-deoxyuridine (EdU) staining for proliferating cells. The techniques of qRT-PCR, Western blot and immunofluorescence were used to detect the mRNA and protein levels of MyoG, MyoD and MHC. The results showed that the expression level of miR-133a increased significantly at the differentiation stage of MDSCs (P<0.01). After overexpression of miR-133a, the positive ratio of proliferating cells significantly decreased (P<0.01), while the gene expression of MyoG (P<0.05) and MHC (P<0.01) significantly increased; The protein expression level of MHC significantly increased (P<0.01) and the number of fused myotubes increased. After miR-133a expression was inhibited, the positive ratio of proliferating cells significantly increased (P<0.01), while the gene expression of MyoG (P<0.01), MyoD (P<0.05) and MHC (P<0.01) significantly decreased; the expression of MHC protein significantly decreased (P<0.01) and the number of fused myotubes decreased. The above results confirmed that miR-133a played a role in inhibiting proliferation and promoting differentiation of MDSCs. The present study enriches the molecular genetic research of bovine muscle growth and development, and provides an important reference for further research on the function of miRNA in regulating animal muscle growth and development.
[1] 代阳. 2016. 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]. 硕士学位论文, 天津农学院, 导师: 丁向彬, pp.12. (Dai Y.2016. Study on the regulation mechanism of microRNA-128 in the proliferation and myogenic difterentiation process of bovine skeletal muscle satellite cells[D]. Thesis for M.S., Tianjin Agricultural University, Supervisor: Ding X B, pp.12.) [2] 李燕, 陈明明, 张俊星, 等. 2019. 牛LncRNA-133a对骨骼肌卫星细胞增殖分化的影响[J]. 中国农业科学, 52(01): 143-153. (Li Y, Chen M M, Zhang J X, et al.2019. Effects of bovine lncRNA-133a on the proliferation and differentiation of skeletal muscle satellite cells[J]. Scientia Agricultura Sinica, 52(01): 143-153.) [3] 孙丽敏, 姜怀志. 2018. mRNA和miRNA调控动物肌肉生长发育及其在绵羊中的研究进展[J]. 中国畜牧杂志, 54(5): 15-24. (Sun L M, Jiang H Z.2018. mRNA and miRNA regulates animal muscle growth and development and the research progress on sheep[J]. Chinese Journal of Animal Science, 54(5): 15-24.) [4] 王亚辉, 王阳, 严云勤, 等. 2014. mir-133b对牛骨骼肌卫星细胞增殖的影响[J]. 黑龙江畜牧兽医,(7): 14-18, 228. (Wang Y H, Wang Y, Yan Y Q, et al.2014. Effect of miRNA-133b (mir-133b) on the proliferation of bovine skeletal muscle satellite cells[J]. Heilongjiang Animal Science and Veterinary Medicine,(7): 14-18, 228.) [5] 朱新峰, 李树峰, 严云勤, 等. 2017. miR-181a对牛骨骼肌卫星细胞分化的影响[J]. 黑龙江畜牧兽医,(11): 19-24, 28, 289. (Zhu X F, LI S F, Yan Y Q, et al.2017. Effect of miR-181a on the differentiation of bovine skeletal muscle satellite cells[J]. Heilongjiang Animal Science and Veterinary Medicine,(11): 19-24, 28, 289.) [6] Bartel D P.2004. MicroRNAs: Genomics, biogenesis, mechanism, and function[J]. Cell, 116(2): 281-297. [7] Buckingham M, Vincent S D.2009. Distinct and dynamic myogenic populations in the vertebrate embryo[J]. Current Opinion in Genetics and Development, 19(5): 444-453. [8] Carthew R W, Sontheimer E J.2009. Origins and mechanisms of miRNAs and siRNAs[J]. Cell, 136(4): 642-655. [9] Chen J F, Mandel E M, Thomson J M, et al.2006. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation[J]. Nature Genetics, 38(2): 228-233. [10] Dai Y, Wang Y M, Zhang W R, et al.2016. The role of microRNA-1 and microRNA-206 in the proliferation and differentiation of bovine skeletal muscle satellite cells[J]. In vitro Cellular & Developmental Biology Animal, 52(01): 27-34. [11] Feng Y, Niu L L, Wei W, et al.2013. A feedback circuit between miR-133 and the ERK1/2 pathway involving an exquisite mechanism for regulating myoblast proliferation and differentiation[J]. Cell Death Disease, 4(11): E934. [12] Gey J, Chen J.2011. microRNAs in skeleltal myogenesis[J]. Cell Cycle, 10(3): 441-448. [13] Hong Q Y D, Xu G L, Hou L J, et al.2020. MicroRNA-22 inhibits proliferation and promotes differentiation of satellite cells in porcine skeletal muscle[J]. Journal of Integrative Agriculture, 19(01): 225-233. [14] Kedde M, Agami R.2008. Interplay between microRNAs and RNA-binding proteins determines developmental processes[J]. Cell Cycle, 7(7): 899-903. [15] Li H, Yang J M, Jiang R, et al.2018. Long non-coding RNA profiling reveals an abundant MDNCR that promotes differentiation of myoblasts by sponging miR-133a[J]. Molecular Therapy-Nucleic Acids, 12: 610-625. [16] Luo W, Nie Q H, Zhang X Q.2013. MicroRNAs involved in skeletal muscle differentiation[J]. Journal of Genetics and Genomics, 40(3): 107-116. [17] Luo Y Q, Wu X X, Ling Z X, et al.2015. microRNA133a targets Foxl2 and promotes differentiation of C2C12 into myogenic progenitor cells[J]. DNA and Cell Biology, 34(01): 29-36. [18] McDaneld T G.2009. MicroRNA: Mechanism of gene regulation and application to livestock[J]. Journal of Animal Science, 87(14): E21-E28. [19] Mok G F, Lozano-Velasco E, Maniou E, et al.2018. miR-133-mediated regulation of the Hedgehog pathway orchestrates embryo myogenesis[J]. Development, 145(12): 159657. [20] Nakasa T, Ishikawa M, Shi M, et al.2010. Acceleration of muscle regeneration by local injection of muscle-specific microRNAs in rat skeletal muscle injury model[J]. Journal of Cellular and Nolecular Medicine, 14(10): 2495-2505. [21] Pallafacchina G, Blaauw B, Schiaffino S.2013. Role of satellite cells in muscle growth and maintenance of muscle mass[J]. Nutrition, Metabolism and Cardiovascular Diseases, 23(Suppl 1):12-18. [22] Pallafacchina G, François S, Regnault B, et al.2010. An adult tissue-specific stem cell in its niche: A gene profiling analysis of in vivo quiescent and activated muscle satellite cells[J]. Stem Cell Research, 4(2): 77-91. [23] Park J K, Liu X, Strauss T J, et al.2007. The miRNA pathway intrinsically controls self-renewal of Drosophila germline stem cells[J]. Current Biology, 6(17): 533-538. [24] Persson P B.2015. Skeletal muscle satellite cells as myogenic progenitors for muscle homoeostasis, growth, regeneration and repair[J]. Acta Physiologica, 213(3): 537-538. [25] Sempere L F, Freemantle S, Pitha-Rowe I, et al.2004. Expression profiling of mammalian microRNAs uncovers a subset of brain expressed microRNAs with possible roles in murine and human neuronal differentiation[J]. Genome Biology, 5(3): 853-858. [26] van Rooij E, Liu N, Olson E N.2008. MicroRNAs flex their muscles[J]. Trends in Genetics, 24(4): 159-166. [27] van Rooij E, Quiat D, Johnson B A, et al.2009. A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance[J]. Developmental Cell, 17(5): 662-673. [28] van Rooij E, Sutherland L B, Qi X, et al.2007. Control of stress-dependent cardiac growth and gene expression by a microRNA[J]. Science, 316(5824): 575-579. [29] Vergara H M, Ramirez J, Rosing T, et al.2018. miR-206 is required for changes in cell adhesion that drive muscle cell morphogenesis in Xenopus laevis[J]. Developmental Biology, 438(2): 94-110. [30] Zhang Z K, Chen Y J, Li B J, et al.2019. Identification of a novel miR-206-Notch3 pathway regulating mouse myoblasts proliferation[J]. Gene, 695: 57-64. [31] Zhang D, Li X, Chen C, et al.2012. Attenuation of p38-mediated miR-1/133 expression facilitates myoblast proliferation during the early stage of muscle regeneration[J]. PLOS ONE, 7(7): e41478. [32] Zhou H B, Xiao J, Wu N, et al.2015. MicroRNA-223 regulates the differentiation and function of intestinal dendritic cells and macrophages by targeting C/EBPβ[J]. Cell Reports, 13(6): 1149-1160.
