FoxO1对牛骨骼肌细胞增殖、凋亡和分化的调控

doi:10.3864/j.issn.0578-1752.2024.06.013

摘要/Abstract

摘要：

【目的】骨骼肌是动物机体的重要组成成分，其生长发育直接影响畜禽肉产量，叉头转录因子O1（forkhead box protein O1, FoxO1）作为重要的转录调控因子，其与骨骼肌生长发育密切相关。探究过表达FoxO1对牛骨骼肌细胞增殖、凋亡与分化的作用，为肉牛遗传改良提供基础材料。【方法】采集牛的多个组织样品，提取其RNA并反转录，利用实时荧光定量PCR（qPCR）构建FoxO1组织表达谱。利用酶消化法分离得到牛骨骼肌细胞，通过观察其分化后肌管的形成以及qPCR检测其分化标志基因的表达情况来检验所分离细胞的分化性能。利用免疫荧光技术对牛骨骼肌细胞进行FoxO1亚细胞定位。设计并包装牛FoxO1过表达腺病毒，以提高牛骨骼肌细胞内FoxO1的表达。利用EdU染色检测过表达FoxO1对细胞相对增殖率的影响。利用流式细胞术检测过表达FoxO1对细胞周期分布的影响。利用qPCR检测过表达FoxO1对牛骨骼肌细胞增殖、凋亡和分化相关基因表达水平的影响。【结果】组织表达谱结果显示FoxO1在多个组织中均有表达，其在成年牛的背脂中表达量最高，在背最长肌组织中表达量最低，且FoxO1在犊牛背最长肌组织中的表达量要极显著高于成年牛的（P<0.01）。亚细胞定位结果显示FoxO1在牛骨骼肌细胞的细胞核和细胞质内均有表达，其细胞核内荧光强度高于细胞质。成功构建FoxO1过表达载体，并完成FoxO1重组过表达腺病毒的包装与扩繁，在感染牛骨骼肌细胞后，能显著提高FoxO1表达水平（P<0.01）。EdU检测显示过表达FoxO1会显著降低细胞增殖率（P<0.01），流式细胞周期检测显示过表达FoxO1会显著增加G1期细胞数并减少S期和G2期细胞数，抑制细胞G1/ S期的转化，并减少G2期细胞的形成。利用qPCR进一步检测发现，增殖相关基因PCNA、CDK1、CDK2、CCNA2、CCNB1、CCND1和CCNE2均极显著下调（P<0.01），促凋亡相关基因BAD和BAX显著上调以及抑凋亡基因BCL2显著下调（P<0.05）。过表达FoxO1导致牛骨骼肌细胞肌管形成量减少，qPCR检测结果发现，骨骼肌分化相关基因MYOD、MYOG、MYF5、MYF6和MYHC的表达量显著下调（P<0.05）。【结论】 FoxO1在牛的不同组织中均有表达，是一个广泛存在的转录调控因子，并且在背最长肌组织生长发育不同阶段存在表达差异，起到阶段调控作用。FoxO1在细胞核和细胞质中均发挥重要的转录调控作用，特别是在细胞核内。过表达FoxO1可能通过抑制细胞增殖相关基因和肌细胞分化相关基因的表达，从而抑制牛骨骼肌细胞的增殖与分化，并且可能通过上调促凋亡基因的表达和下调抑凋亡基因的表达来促使牛骨骼肌细胞凋亡的发生。

关键词: FoxO1, 牛骨骼肌细胞, 增殖, 分化

Abstract:

【Objective】 Skeletal muscle is an important component of animal body, and its growth and development directly affect the meat yield of livestock and poultry. As an important transcription regulator, fork head box protein O1 (FoxO1) is closely related to the growth and development of skeletal muscle. Exploring the effects of overexpression of FoxO1 gene on the proliferation, apoptosis and differentiation of bovine skeletal muscle cells could provide the theoretical basis for genetic improvement of beef. 【Method】 The tissue expression profile of FoxO1 was constructed by real-time fluorescence quantitative PCR (qPCR). To test the cell differentiation performance of bovine skeletal muscle cells which were isolated by enzyme digestion, the myotubes formation was observed and the expressions of differentiation marker genes were detected by qPCR. The subcellular localization of bovine skeletal muscle cells was carried out by immunofluorescence technique. In order to improve the expression of FoxO1 in bovine skeletal muscle cells, bovine FoxO1 overexpression adenovirus were designed and packaged. The effect of FoxO1 overexpression on the relative proliferation rate of cells was detected by EdU staining. Flow cytometry was used to detect the effect of FoxO1 overexpression on cell cycle distribution. The effects of FoxO1 overexpression on the expression levels of genes related to proliferation, apoptosis and differentiation of bovine skeletal muscle cells were investigated by qPCR.【Result】 The results of tissue expression spectrum showed that FoxO1 was expressed in many tissues, with the highest expression in the back fat of adult cattle and the lowest expression in the longissimus dorsi muscle tissue, while the expression of FoxO1 in the longissimus dorsi muscle tissue of calves was significantly higher than that of adult cattle (P<0.01). The results of subcellular localization showed that FoxO1 was expressed in the nucleus and cytoplasm of bovine skeletal muscle cells, and the fluorescence intensity in the nucleus was higher than that in the cytoplasm. The FoxO1 overexpression vector was successfully constructed, and the packaging and propagation of FoxO1 recombinant adenovirus were completed. After infecting bovine skeletal muscle cells, the FoxO1 expression level was significantly improved (P<0.01). EdU detection showed that overexpression of FoxO1 significantly reduced the cell proliferation rate (P<0.01), and flow cytometry showed that overexpression of FoxO1 significantly increased the number of cells in G1 phase and decreased the number of cells in S phase and G2 phase, inhibited the transformation of cells in G1/S phase and reduced the formation of cells in G2 phase. Further detection by qPCR found that the proliferation-related genes of PCNA, CDK1, CDK2, CCNA2, CCNB1, CCND1 and CCNE2 were significantly down-regulated (P<0.01), the apoptosis-related genes BAD and BAX were significantly up-regulated, and the apoptosis-inhibiting gene BCL2 was significantly down-regulated (P<0.05). After FoxO1 was overexpressed, the myotube formation of bovine skeletal muscle cells decreased, and the expression levels of MYOD, MYOG, MYF5, MYF6 and MYHC related to skeletal muscle differentiation were significantly decreased by qPCR detection (P<0.05). 【Conclusion】 FoxO1 was expressed in different tissues of cattle, and it was a ubiquitous transcription regulator. There were differences in expression at different stages of the growth and development of longissimus dorsi muscle, which played a role in stage regulation. FoxO1 played a transcriptional regulatory role in both the nucleus and cytoplasm, especially in the nucleus. Overexpression of FoxO1 might inhibit the proliferation and differentiation of bovine skeletal muscle cells by inhibiting the expression of genes related to cell proliferation and muscle cell differentiation, and might promote the apoptosis of bovine skeletal muscle cells by up-regulating the expression of apoptosis-promoting genes and down-regulating the expression of apoptosis- inhibiting genes.

Key words: FoxO1, bovine skeletal muscle cells, proliferation, differentiation

姜超, 张久盘, 宋雅萍, 宋小雨, 吴昊, 魏大为. FoxO1对牛骨骼肌细胞增殖、凋亡和分化的调控[J]. 中国农业科学, 2024, 57(6): 1191-1203.

JIANG Chao, ZHANG JiuPan, SONG YaPing, SONG XiaoYu, WU Hao, WEI DaWei. Study on the Role of FoxO1 in Regulating the Proliferation, Apoptosis and Differentiation of Bovine Skeletal Muscle Cells[J]. Scientia Agricultura Sinica, 2024, 57(6): 1191-1203.

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.0578-1752.2024.06.013

https://www.chinaagrisci.com/CN/Y2024/V57/I6/1191

图/表 12

表1

表2

图1

图2

图3

图4

图5

图6

图7

图8

图9

图10

参考文献 31

[1]	张天留, 葛菲, 朱波, 高会江, 李俊雅, 高雪. 肉牛种业科技创新发展现状与趋势分析. 中国畜禽种业, 2022, 18(10): 5-16.
	ZHANG T L, GE F, ZHU B, GAO H J, LI J Y, GAO X. Present situation and trend analysis of scientific and technological innovation in beef cattle seed industry. The Chinese Livestock and Poultry Breeding, 2022, 18(10): 5-16. (in Chinese)
[2]	付玉, 张博, 凌遥, 张浩. 骨骼肌生长发育过程及调控研究现状. 中国畜牧兽医, 2021, 48(10): 3565-3574. doi: 10.16431/j.cnki.1671-7236.2021.10.007
	FU Y, ZHANG B, LING Y, ZHANG H. Reviews on process and regulation of skeletal muscle growth and development. China Animal Husbandry & Veterinary Medicine, 2021, 48(10): 3565-3574. (in Chinese)
[3]	AHMAD S S, CHUN H J, AHMAD K, SHAIKH S, LIM J H, ALI S, HAN S S, HUR S J, SOHN J H, LEE E J, CHOI I. The roles of growth factors and hormones in the regulation of muscle satellite cells for cultured meat production. Journal of Animal Science and Technology, 2023, 65(1): 16-31. doi: 10.5187/jast.2022.e114 pmid: 37093925
[4]	MIERZEJEWSKI B, GRABOWSKA I, JACKOWSKI D, IRHASHAVA A, MICHALSKA Z, STREMIŃSKA W, JAŃCZYK-ILACH K, CIEMERYCH M A, BRZOSKA E. Mouse CD146+ muscle interstitial progenitor cells differ from satellite cells and present myogenic potential. Stem Cell Research & Therapy, 2020, 11(1): 341.
[5]	MOHAMMADABADI M, BORDBAR F, JENSEN J, DU M, GUO W. Key genes regulating skeletal muscle development and growth in farm animals. Animals, 2021, 11(3): 835. doi: 10.3390/ani11030835
[6]	ESTEVES DE LIMA J, RELAIX F. Master regulators of skeletal muscle lineage development and pluripotent stem cells differentiation. Cell Regeneration, 2021, 10(1): 31. doi: 10.1186/s13619-021-00093-5
[7]	ZAMMIT P S. Function of the myogenic regulatory factors Myf5, MyoD, Myogenin and MRF₄ in skeletal muscle, satellite cells and regenerative myogenesis. Seminars in Cell & Developmental Biology, 2017, 72: 19-32.
[8]	SHIRAKAWA T, TOYONO T, INOUE A, MATSUBARA T, KAWAMOTO T, KOKABU S. Factors regulating or regulated by myogenic regulatory factors in skeletal muscle stem cells. Cells, 2022, 11(9): 1493. doi: 10.3390/cells11091493
[9]	侯任达, 张润, 侯欣华, 王立贤, 张龙超. 畜禽肌纤维发育规律及相关基因研究进展. 畜牧兽医学报, 2022, 53(10): 3279-3286.
	HOU R D, ZHANG R, HOU X H, WANG L X, ZHANG L C. Research progress on the pattern of muscle fiber development and related genes in livestock and poultry. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3279-3286. (in Chinese)
[10]	束婧婷, 姬改革, 单艳菊, 章明, 肖芹, 屠云洁, 盛中伟, 张笛, 邹剑敏. 基于表达谱芯片挖掘鸡骨骼肌不同类型肌纤维的差异表达基因. 中国农业科学, 2017, 50(14): 2826-2836. doi:10.3864/j.issn.0578-1752.2017.14.018.
	SHU J T, JI G G, SHAN Y J, ZHANG M, XIAO Q, TU Y J, SHENG Z W, ZHANG D, ZOU J M. Analysis of differential expression genes between different myofiber types in chicken skeletal muscle based on gene expression microarray. Scientia Agricultura Sinica, 2017, 50(14): 2826-2836. doi:10.3864/j.issn.0578-1752.2017.14.018. (in Chinese)
[11]	李燕, 陈明明, 张俊星, 张林林, 李新, 郭宏, 丁向彬, 刘新峰. 牛LncRNA-133a对骨骼肌卫星细胞增殖分化的影响. 中国农业科学, 2019, 52(1): 143-153. doi: 10.3864/j.issn.0578-1752.2019.01.013.
	LI Y, CHEN M M, ZHANG J X, ZHANG L L, LI X, GUO H, DING X B, LIU X F. Effects of bovine LncRNA-133a on the proliferation and differentiation of skeletal muscle satellite cells. Scientia Agricultura Sinica, 2019, 52(1): 143-153. doi: 10.3864/j.issn.0578-1752.2019.01.013. (in Chinese)
[12]	CALNAN D R, BRUNET A. The FoxO code. Oncogene, 2008, 27(16): 2276-2288. doi: 10.1038/onc.2008.21 pmid: 18391970
[13]	GUO S C, MANGAL R, DANDU C T, GENG X K, DING Y C. Role of forkhead box protein O1 (FoxO1) in stroke: a literature review. Aging and Disease, 2022, 13(2): 521-533. doi: 10.14336/AD.2021.0826
[14]	YAMASHITA A, HATAZAWA Y, HIROSE Y, ONO Y, KAMEI Y. FOXO 1 delays skeletal muscle regeneration and suppresses myoblast proliferation. Bioscience, Biotechnology, and Biochemistry, 2016, 80(8): 1531-1535. doi: 10.1080/09168451.2016.1164585
[15]	ZHANG H, CHI M Y, CHEN L L, SUN X P, WAN L L, YANG Q J, GUO C. Daidzein alleviates cisplatin-induced muscle atrophy by regulating Glut4/AMPK/FoxO pathway. Phytotherapy Research, 2021, 35(8): 4363-4376. doi: 10.1002/ptr.7132 pmid: 33876509
[16]	WANG B, ZHANG A Q, WANG H D, KLEIN J D, TAN L, WANG Z M, DU J, NAQVI N, LIU B C, WANG X H. miR-26a limits muscle wasting and cardiac fibrosis through exosome-mediated microRNA transfer in chronic kidney disease. Theranostics, 2019, 9(7): 1864-1877. doi: 10.7150/thno.29579 pmid: 31037144
[17]	RU W X, LIU K P, YANG J M, LIU J Y, QI X L, HUANG B Z, CHEN H. miR-183/96/ 182 cluster regulates the development of bovine myoblasts through targeting FoxO1. Animals, 2022, 12(20): 2799. doi: 10.3390/ani12202799
[18]	ZHANG X J, JIANG L S, LIU H M. Forkhead box protein O1: functional diversity and post-translational modification, a new therapeutic target? Drug Design, Development and Therapy, 2021, 15: 1851-1860. doi: 10.2147/DDDT.S305016 pmid: 33976536
[19]	LIU Y, WANG Y, SHAN T, GUO J, XU C, LIU J. The tissue-specific and developmental expressionpatterns of the forkhead transcription factor FoxO1gene in pigs. Journal of Animal and Feed Sciences, 2008, 17(2): 182-190. doi: 10.22358/jafs/66598/2008
[20]	PANG W J, YU T Y, BAI L, YANG Y J, YANG G S. Tissue expression of porcine FoxO1 and its negative regulation during primary preadipocyte differentiation. Molecular Biology Reports, 2009, 36(1): 165-176. doi: 10.1007/s11033-007-9163-6
[21]	YOSHIMOCHI K, DAITOKU H, FUKAMIZU A. PCAF represses transactivation function of FOXO1 in an acetyltransferase-independent manner. Journal of Receptor and Signal Transduction Research, 2010, 30(1): 43-49. doi: 10.3109/10799890903517947 pmid: 20041807
[22]	WU Y J, FANG Y H, CHI H C, CHANG L C, CHUNG S Y, HUANG W C, WANG X W, LEE K W, CHEN S L. Insulin and LiCl synergistically rescue myogenic differentiation of FoxO1 over- expressed myoblasts. PLoS ONE, 2014, 9(2): e88450. doi: 10.1371/journal.pone.0088450
[23]	SCHACHTER T N, SHEN T S, LIU Y W, SCHNEIDER M F. Kinetics of nuclear-cytoplasmic translocation of Foxo1 and Foxo3A in adult skeletal muscle fibers. American Journal of Physiology Cell Physiology, 2012, 303(9): C977-C990. doi: 10.1152/ajpcell.00027.2012
[24]	CHEN J Y, LU Y, TIAN M Y, HUANG Q R. Molecular mechanisms of FOXO1 in adipocyte differentiation. Journal of Molecular Endocrinology, 2019, 62(3): R239-R253. doi: 10.1530/JME-18-0178
[25]	MONSALVE M, OLMOS Y. The complex biology of FOXO. Current Drug Targets, 2011, 12(9): 1322-1350. doi: 10.2174/138945011796150307 pmid: 21443460
[26]	LIU F, QU R F, YANG L M, SHI G, HAO S H, HU C M. Circular RNA controls tumor occurrence and development via cell cycle regulation. OncoTargets and Therapy, 2022, 15: 993-1009. doi: 10.2147/OTT.S371629 pmid: 36134387
[27]	HUANG H J, TINDALL D J. CDK2 and FOXO1: a fork in the road for cell fate decisions. Cell Cycle, 2007, 6(8): 902-906. pmid: 17457058
[28]	GEBREMEDHN S, SALILEW-WONDIM D, HOELKER M, RINGS F, NEUHOFF C, THOLEN E, SCHELLANDER K, TESFAYE D. microRNA-183-96-182 cluster regulates bovine granulosa cell proliferation and cell cycle transition by coordinately targeting FOXO1. Biology of Reproduction, 2016, 94(6): 127, 1-11.
[29]	CIFARELLI V, LEE S, KIM D H, ZHANG T, KAMAGATE A, SLUSHER S, BERTERA S, LUPPI P, TRUCCO M, DONG H H. FOXO 1 mediates the autocrine effect of endothelin-1 on endothelial cell survival. Molecular Endocrinology, 2012, 26(7): 1213-1224. doi: 10.1210/me.2011-1276
[30]	FERRI P, BARBIERI E, BURATTINI S, GUESCINI M, D'EMILIO A, BIAGIOTTI L, DEL GRANDE P, DE LUCA A, STOCCHI V, FALCIERI E. Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts. Journal of Cellular Biochemistry, 2009, 108(6): 1302-1317. doi: 10.1002/jcb.22360 pmid: 19830700
[31]	来裕婷, 朱菲菲, 王轶敏, 郭宏, 张林林, 李新, 郭益文, 丁向彬. PSMB5蛋白对牛骨骼肌卫星细胞增殖与成肌分化的影响. 中国农业科学, 2020, 53(20): 4287-4296. doi: 10.3864/j.issn.0578-1752.2020.20.016.
	LAI Y T, ZHU F F, WANG Y M, GUO H, ZHANG L L, LI X, GUO Y W, DING X B. Effects of PSMB5 on the proliferation and myogenic differentiation of skeletal muscle satellite cells. Scientia Agricultura Sinica, 2020, 53(20): 4287-4296. doi: 10.3864/j.issn.0578-1752.2020.20.016. (in Chinese)

基因名称 Gene name	引物序列 Primer sequence (5′→3′)	产物长度 Products length (bp)	基因登录号 Genbank ID
GAPDH	F：CCAACGTGTCTGTTGTGGAT	80	NM_001034034
GAPDH	R：CTGCTTCACCACCTTCTTGA	80	NM_001034034
FoxO1	F：GCAGATTTACGAGTGGATGG	86	XM_025000053.1
FoxO1	R：TTGAATTCTTCCAGCCCG	86	XM_025000053.1
PCNA	F：GAACCTCACCAGCATGTCCA	97	NM_001034494
PCNA	R：TACTAGTGCCAACGTGTCCG	97	NM_001034494
CDK1	F：GAAGGGGTTCCTAGTACTGC	176	NM_174016
CDK1	R：ATGAACTGACCAGGAGGG	176	NM_174016
CDK2	F：GCCAGGAGTTACTTCTATGC	102	NM_001014934
CDK2	R：CTCCGTCCATCTTCATCC	102	NM_001014934
CCNA2	F：ACACAGTCACAGGACAAAGC	107	NM_001075123
CCNA2	R：TCTGAGGTAGGTCTGGTGAA	107	NM_001075123
CCNB1	F：TGGAGAGGTTGATGTTGAGC	95	NM_001045872
CCNB1	R：TCTGAGAAGGAGGAAAGTGC	95	NM_001045872
CCND1	F：TCCTCTCCTATCACCGCCTGAC	175	NM_001046273
CCND1	R：ACCTCCTCCTCCTCCTCTTCCT	175	NM_001046273
CCNE2	F：GCTTATGTCACTGATGGTGCTTG	112	NM_001015665
CCNE2	R：TTAGCCAGGAGATGACCGTTAC	112	NM_001015665
MYOD	F：GGCCGCTGTTTACTGTGGG	162	NM_001040478
MYOD	R：CAGCCGCTGGTTTGGGTT	162	NM_001040478
MYOG	F：CTATGACGGGGAGAACTACCTG	249	NM_001111325
MYOG	R：CATTCACCTTCTTGAGTCTGCG	249	NM_001111325
MYHC	F：GCCCACTTCTCCCTCATTCACT	201	XM_010815980
MYHC	R：ACCCTTCTTCTTGCCACCTTTC	201	XM_010815980
MYF5	F：CTCTGATGGCATGCCTGAATGT	180	NM_174116
MYF5	R：GGCAATCCAGGTTGCTCTGA	180	NM_174116
MYF6	F：TGGACCCCTTCAGCTACAGAC	126	NM_181811
MYF6	R：CTTCCTTGGCAGTTATCACGAG	126	NM_181811
BAX	F：GAGATGAATTGGACAGTAACA	118	NM_173894
BAX	R：TTGAAGTTGCCGTCAGAA	118	NM_173894
BAD	F：CTCAGCAAGCACTGGCTAACA	270	NM_001035459
BAD	R：GTGAAACTCGTCGCTCATCCT	270	NM_001035459
BCL2	F：TTAGCCAGTGCTTGCTGAGAC	86	NM_001166486
BCL2	R：TTAGCCAGTGCTTGCTGAGAC	86	NM_001166486

基因名称 Gene name	引物方向 Primer direction	引物序列 Primer sequence (5′→3′)	产物长度 Products length (bp)	基因登录号 Genbank ID
FoxO1	F-Kpn I-FoxO1	CGGGGTACCGCCACCATGGCCGAAGCGCCCCAGGTG	1998	XM_025000053.1
FoxO1	R-Hind IlI-FoxO1	CCCAAGCTTTCAGCCTGACACCCAGCTGTGTG	1998	XM_025000053.1