藏鸡胚胎不同发育阶段腿肌组织中miRNA的分析及鉴定

doi:10.16431/j.cnki.1671-7236.2025.04.021

摘要/Abstract

摘要： 【目的】探索藏鸡胚胎期与骨骼肌生长发育相关的微小RNA (miRNA)，对其进行差异表达分析并构建miRNA-mRNA调控网络，为进一步了解miRNA在鸡肌肉生长发育过程中的调控机制提供理论基础。【方法】采集藏鸡胚胎期第10(E10)、14(E14)、18(E18)天腿肌组织进行转录组测序。通过生物信息学分析筛选出在3个日龄中均差异表达的miRNA，对差异表达的miRNA进行GO功能与KEGG通路富集分析，筛选与肌肉发育相关的mRNA和miRNA构建mRNA-miRNA调控网络，并随机选择6个差异表达的miRNAs进行实时荧光定量PCR验证。【结果】共有52个miRNAs在E10和E14中差异表达，137个miRNAs在E10和E18中差异表达，33个miRNAs在E14和E18中差异表达，有8个miRNAs在3个藏鸡不同时期胚胎腿肌中均差异表达。GO功能富集结果显示，与骨骼肌生长发育相关的条目有肌动蛋白结合、肌原纤维的组装、肌动球蛋白结构组织和基于肌动蛋白丝的过程、肌纤维膜等。KEGG通路富集结果发现，大量基因显著富集到与细胞的黏附、生存、迁移、增殖、代谢和分化、免疫调节等生物学功能有关的信号通路中。miRNA-mRNA调控网络结果显示，miRNA-210a-3p-MYO1D/PDLIM3、miRNA-1662-DMD/SYNM、miRNA-184-3p-FHOD1等相互调控可能在鸡胚胎期肌肉生长发育过程中起重要作用。研究筛选出miRNA-133c-3p、miRNA-184-3p、miRNA-1662、miRNA-210a-3p、miRNA-1a-3p等miRNAs可能参与肌肉生长发育的过程。实时荧光定量PCR验证结果显示，6个miRNAs的表达趋势与转录组测序结果相一致。【结论】本研究共测得222个差异表达的miRNAs，其中有8个miRNAs在3个日龄中均差异表达，为后续miRNA在调控鸡胚胎时期肌肉生长发育的研究提供理论依据。

关键词: 藏鸡; 胚胎期; 肌肉发育; miRNA; 转录组测序

Abstract: 【Objective】 This study was aimed to explore microRNA (miRNA) related to skeletal muscle growth and development during the embryonic period of Tibetan chickens,analyze their differential expression and construct the miRNA-mRNA regulatory network,so as to provide a theoretical basis for further understanding the regulatory mechanism of miRNA in the growth and development of chicken muscles.【Method】 RNA-Seq was performed on the leg muscle tissues of Tibetan chickens at the 10th (E10),14th (E14) and 18th (E18) days of embryonic stage.The differentially expressed miRNA among three periods was screened through bioinformatics analysis,and the differentially expressed miRNA was enriched by GO function and KEGG pathway,and the mRNA and miRNA related to muscle development were screened to construct the mRNA-miRNA regulatory network.Six differentially expressed miRNAs were randomly selected for Real-time quantitative PCR to verify RNA-Seq results.【Result】 The results showed that 52 miRNAs were differentially expressed between E10 and E14,137 miRNAs were differentially expressed between E10 and E18,33 miRNAs were differentially expressed between E14 and E18,and 8 miRNAs were differentially expressed in leg muscles of Tibetan chickens among three periods.The results of GO function enrichment analysis showed that the items related to skeletal muscle growth and development were actin binding,myofibrillar assembly,actomyosin structure and actin filament-based processes,muscle fiber membrane,etc.KEGG pathway enrichment analysis showed that a large number of genes were significantly enriched in the signaling pathways related to the biological functions of cell adhesion,survival,migration,proliferation,metabolism and differentiation,and immune regulation.The results of miRNA-3P-MYO1D/PDLIM3,miRNA-1662-DMD/SYNM,and miRNA-184-3p-FHOD1 might play an important role in the muscle growth and development of chicken embryos.miRNAs such as miRNA-133c-3p,miRNA-184-3p,miRNA-1662,miRNA-210a-3p and miRNA-1a-3p might be involved in the process of muscle growth and development.Real-time quantitative PCR results showed that the expression of six miRNAs were consistent with RNA-Seq results.【Conclusion】 A total of 222 differentially expressed miRNAs were detected,among which 8 miRNAs were differentially expressed at all three periods,providing theoretical basis for subsequent studies on the regulation of miRNA on muscle growth and development in chicken embryos.

Key words: Tibetan chickens; embryonic period; muscle development; miRNA; RNA-Seq

中图分类号:

S831.2

李志毅, 李洁, 陈楚雯, 农意, 王佳燕, 王孜, 吴锦波, 李志雄. 藏鸡胚胎不同发育阶段腿肌组织中miRNA的分析及鉴定[J]. 中国畜牧兽医, 2025, 52(4): 1681-1693.

LI Zhiyi, LI Jie, CHEN Chuwen, NONG Yi, WANG Jiayan, WANG Zi, WU Jinbo, LI Zhixiong. Analysis and Identification of miRNA in Leg Muscle Tissue of Tibetan Chicken Embryos at Different Developmental Stages[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(4): 1681-1693.

参考文献

[1] 陈雪娇,钟海安,张博,等.藏鸡高原低氧适应性微进化机制研究进展[J].中国畜牧杂志,2023,59(2):1-5.CHEN X J,ZHONG H A,ZHANG B,et al.Research progress on microevolution mechanism of high-altitude hypoxic adaptation in Tibetan chicken[J].Chinese Journal of Animal Science,2023,59(2):1-5.(in Chinese)
[2] 纪韦韦,徐银,洪文龙.雪山草鸡氨基酸组成与营养价值研究[J].安徽农业科学,2012,40(21):10919-10921.JI W W,XU Y,HONG W L.Study on the amino acid composition and nutritional value of Xueshan chicken[J].Journal of Anhui Agricultural Sciences,2012,40(21):10919-10921.(in Chinese)
[3] BRAUN T,GAUTEL M.Transcriptional mechanisms regulating skeletal muscle differentiation,growth and homeostasis[J].Nature Reviews Molecular Cell Biology,2011,12(6):349-361.
[4] BUCKINGHAM M.Gene regulatory networks and cell lineages that underlie the formation of skeletal muscle[J].Proceedings of the National Academy of Sciences of the United States of America,2017,114(23):5830-5837.
[5] 闫俊书,周维仁,宦海林,等.家禽肌纤维的生长发育规律及其调控[J].江苏农业科学,2010,5:276-279.YAN J S,ZHOU W R,HUAN H L,et al.The growth traits and regulation of myofiber in poultry[J].Jiangsu Agricultural Sciences,2010,5:276-279.(in Chinese)
[6] 宋陈玲,姚婕,王继文.鸟类胚胎期肌肉发育的影响因素分析[J].中国家禽,2012,34(21):54-55.SONG C L,YAO J,WANG J W.Analysis of influencing factors of muscle development in bird embryo[J].China Poultry,2012,34(21):54-55.(in Chinese)
[7] LIU J,LI F,HU X,et al.Deciphering the miRNA transcriptome of breast muscle from the embryonic to post-hatching periods in chickens[J].BMC Genomics,2021,22(1):64.
[8] BARTEL D P.microRNAs:Target recognition and regulatory functions[J].Cell,2009,136(2):215-233.
[9] XU Z,LIU Q,NING C,et al.miRNA profiling of chicken follicles during follicular development[J].Scientific Reports,2024,14(1):2212.
[10] 丛百林,薛吕帆,盖凯,等.利用miRNA测序技术鉴定调控种公鸡精子发生的候选基因[J].华北农学报,2022,37(S1):333-338.CONG B L,XUE L F,GAI K,et al.Identification of candidate genes regulating spermatogenesis in breeder cocks by miRNA sequencing[J].Acta Agriculturae Boreali-Sinica,2022,37(S1):333-338.(in Chinese)
[11] O'ROURKE J R,GEORGES S A,SEAY H R,et al.Essential role for dicer during skeletal muscle development[J].Developmental Biology,2007,311(2):359-368.
[12] ZHAO J,ZHAO X,SHEN X,et al.circCCDC91 regulates chicken skeletal muscle development by sponging miR-15 family via activating IGF1-PI3K/Akt signaling pathway[J].Poultry Science,2022,101(5):101803.
[13] DEY P,SOYER M,DEY B.microRNA-24-3p promotes skeletal muscle differentiation and regeneration by regulating HMGA1[J].Cellular and Molecular Life Sciences,2022,79(3):170.
[14] YUAN C,XIE H,CHEN X,et al.Roles of miR-196a and miR-196b in zebrafish motor function[J].Biomolecules,2023,13(3):554.
[15] WANG X Y,CHEN X L,HUANG Z Q,et al.microRNA-499-5p regulates porcine myofiber specification by controlling Sox6 expression[J].Animal,2017,11(12):2268-2274.
[16] ZHANG Z,DENG K,KANG Z,et al.microRNA profiling reveals miR-145-5p inhibits goat myoblast differentiation by targeting the coding domain sequence of USP13[J].Federation of American Societies for Experimental Biology Journal,2022,36(7):e22370.
[17] ALI A,MURANI E,HADLICH F,et al.Prenatal skeletal muscle transcriptome analysis reveals novel microRNA-mRNA networks associated with intrauterine growth restriction in pigs[J].Cells,2021,10(5):1007.
[18] HE M,ZHANG W,WANG S,et al.microRNA-181a regulates the proliferation and differentiation of Hu sheep skeletal muscle satellite cells and targets the YAP1 gene[J].Genes (Basel),2022,13(3):520.
[19] WANG J,YANG L Z,ZHANG J S,et al.Effects of microRNAs on skeletal muscle development[J].Gene,2018,668:107-113.
[20] RAZA S H A,KASTER N,KHAN R,et al.The role of microRNAs in muscle tissue development in beef cattle[J].Genes (Basel),2020,11(3):295.
[21] SHINTANI-ISHIDA K,TSURUMI R,IKEGAYA H.Decrease in the expression of muscle-specific miRNAs,miR-133a and miR-1,in myoblasts with replicative senescence[J].PLoS One,2023,18(1):e0280527.
[22] CHEN J F,MANDEL E M,THOMSON J M,et al.The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation[J].Nature Genetics,2006,38(2):228-233.
[23] LIU N,BEZPROZVANNAYA S,WILLIAMS A H,et al.microRNA-133a regulates cardiomyocyte proliferation and suppresses smooth muscle gene expression in the heart[J].Genes and Development,2008,22(23):3242-3254.
[24] FAN X,XING Y,LIU L,et al.Screening of microRNAs with potential systemic effects released from goose fatty liver[J].Journal of Poultry Science,2021,58(4):263-269.
[25] MIAO X,LIU L,LIU L,et al.Regulation of mRNA and miRNA in the response to Salmonella enterica serovar enteritidis infection in chicken cecum[J].Biomed Central Veterinary Research,2022,18(1):437.
[26] CHAN Y C,BANERJEE J,CHOI S Y,et al.miR-210:The master hypoxamir[J].Microcirculation,2012,19(3):215-223.
[27] YANG X,SHI L,YI C,et al.miR-210-3p inhibits the tumor growth and metastasis of bladder cancer via targeting fibroblast growth factor receptor-like 1[J].American Journal of Cancer Research,2017,7(8):1738-1753.
[28] EVANGELISTA A F,OLIVEIRA R J,VA O S,et al.Integrated analysis of mRNA and miRNA profiles revealed the role of miR-193 and miR-210 as potential regulatory biomarkers in different molecular subtypes of breast cancer[J].BMC Cancer,2021,21(1):76.
[29] CHEN Q,ZHANG H,ZHANG J,et al.miR-210-3p promotes lung cancer development and progression by modulating USF1 and PCGF3[J].OncoTargets and Therapy,2021,14:3687-3700.
[30] CICCHILLITTI L,DI STEFANO V,ISAIA E,et al.Hypoxia-inducible factor 1-α induces miR-210 in normoxic differentiating myoblasts[J].Journal of Biological Chemistry,2012,287(53):44761-44771.
[31] MUTHARASAN R K,NAGPAL V,ICHIKAWA Y,et al.microRNA-210 is upregulated in hypoxic cardiomyocytes through Akt-and p53-dependent pathways and exerts cytoprotective effects[J].American Journal of Physiology Heart and Circulatory Physiology,2011,301(4):H1519-H1530.
[32] CHAN S Y,ZHANG Y Y,HEMANN C,et al.microRNA-210 controls mitochondrial metabolism during hypoxia by repressing the iron-sulfur cluster assembly proteins ISCU1/2[J].Cell Metabolism,2009,10(4):273-284.
[33] ZOU J F,WU X N,SHI R H,et al.Inhibition of microRNA-184 reduces H2O2-mediated cardiomyocyte injury via targeting FBXO28[J].European Review for Medical and Pharmacological Sciences,2020,24(21):11251-11258.
[34] HUANG J,LI X,LI H,et al.Down-regulation of microRNA-184 contributes to the development of cyanotic congenital heart diseases[J].International Journal of Clinical and Experimental Pathology,2015,8(11):14221-14227.
[35] GURHA P,CHEN X,LOMBARDI R,et al.Knockdown of plakophilin 2 downregulates miR-184 through CpG hypermethylation and suppression of the E2F1 pathway and leads to enhanced adipogenesis in vitro[J].Circulation Research,2016,119(6):731-750.
[36] SANGER J W,WANG J,FAN Y,et al.Assembly and maintenance of myofibrils in striated muscle[J].Handbook of Experimental Pharmacology,2017,235:39-75.
[37] BOUJU S,PIÉTU G,LE CUNFF M,et al.Exclusion of muscle specific actinin-associated LIM protein (ALP) gene from 4q35 facioscapulohumeral muscular dystrophy (FSHD) candidate genes[J].Neuromuscular Disorders,1999,9(1):3-10.
[38] YIN H,ZHAO J,HE H,et al.gga-miR-3525 targets PDLIM3 through the MAPK signaling pathway to regulate the proliferation and differentiation of skeletal muscle satellite cells[J].International Journal of Molecular Sciences,2020,21(15):5573.
[39] YANG Y.Skeletal morphogenesis during embryonic development[J].Critical Reviews in Eukaryotic Gene Expression,2009,19(3):197-218.
[40] MATSUOKA K,PARK K A,ITO M,et al.Osteoclast-derived complement component 3a stimulates osteoblast differentiation[J].Journal of Bone and Mineral Research,2014,29(7):1522-1530.
[41] VAFIADAKI E,ARVANITIS D A,SANOUDOU D.Muscle LIM protein:Master regulator of cardiac and skeletal muscle functions[J].Gene,2015,566(1):1-7.
[42] KONG Y,FLICK M J,KUDLA A J,et al.Muscle LIM protein promotes myogenesis by enhancing the activity of MyoD[J].Molecular And Cellular Biology,1997,17(8):4750-4760.
[43] CUI C,HAN S,TANG S,et al.The autophagy regulatory molecule CSRP3 interacts with LC3 and protects against muscular dystrophy[J].International Journal of Molecular Sciences,2020,21(3):749.
[44] XIONG Y,WANG Y,XU Q,et al.LKB1 regulates goat intramuscular adipogenesis through focal adhesion pathway[J].Frontiers in Physiology,2021,12:755598.
[45] HUANG H,LIU L,LI C,et al.Fat mass and obesity-associated (FTO) gene promoted myoblast differentiation through the focal adhesion pathway in chicken[J].3 Biotech,2020,10(9):403.
[46] LI Y,CHEN Y,LIAO Y,et al.Photobiomodulation therapy moderates cancer cachexia-associated muscle wasting through activating PI3K/Akt/FoxO3a pathway[J].Apoptosis,2024,29(5-6):663-680.
[47] YU H,ZHU G,WANG D,et al.PI3K/Akt/FOXO3a pathway induces muscle atrophy by ubiquitin-proteasome system and autophagy system in COPD rat model[J].Cell Biochem Biophys,2024,82(2):805-815.
[48] KIMMICH M J,SUNDARAMURTHY S,GEARY M A,et al.FHOD-1/profilin-mediated actin assembly protects sarcomeres against contraction-induced deformation in C.elegans[J].BioRxiv,2024,3:582848.