鸡胚胎期肌纤维形成相关miRNAs的筛选与分析

Screening and Analysis of miRNAs Related to Chicken Embryonic Muscle Fiber Formation

研究旨在挖掘鸡胚胎期肌纤维形成相关miRNAs,为进一步探究miRNA在肌肉发育过程中的调控机制提供理论基础。研究采集玫瑰冠鸡、科宝鸡胚胎期第8天(E8)和第14天(E14)腿肌组织进行HE染色和small RNA测序,通过生物信息学分析差异表达miRNAs,随机选择14个差异表达的miRNAs进行荧光定量PCR(qRT-PCR)验证结果的准确性。结果显示,玫瑰冠鸡、科宝鸡E8的肌纤维直径和横截面积极显著小于E14(P<0.01),E8的肌纤维密度极显著高于E14(P<0.01)。通过生物信息学分析,在12个测序文库中共鉴定出2455个已知miRNAs和329个新的miRNAs,其中在E8和E14(C8TvsC14T_R8TvsR14T)中筛选出499个差异表达miR-NAs。GO富集结果显示,差异miRNA靶基因在细胞迁移、代谢过程、细胞增殖、细胞发育和小分子结合等GO条目中显著富集。KEGG富集分析发现,大量的靶基因富集到与细胞周转和代谢相关的信号通路中,同时富集到TGF-β、Notch、Hedgehog、Wnt、FoxO等与肌肉发育相关的信号通路。miRNA-mRNA联合分析结果表明,miR-3594-3p-APOBEC2、miR-1635-FHL3/MEF2D、miR-193-3p-HDAC3、miR-1724-PRKAG3、miR-1698-PRKAG3等互作对可能是调控鸡胚胎期肌纤维形成和肌肉发育的重要因子。研究表明,鸡胚胎期肌纤维形成受到多种miRNAs和信号通路的调控,筛选出miR-3594-3p、miR-1635、miR-193-3p、miR-1724、miR-1698等miRNAs可能在肌纤维形成过程中发挥重要作用。

This study aimed to explore the miRNAs related to chicken embryonic muscle fiber formation,and to provide a theoretical basis for further exploring the regulatory mechanism of miRNAs in muscle development.In this study,small RNA sequencing was performed on leg muscle tissues of Rose-crown chicken and Cobb chicken on embryonic day 8(E8)and day 14(E14).Differentially expressed miRNAs were analyzed by bioinformatics.Fourteen differentially expressed miRNAs were randomly selected for fluorescence quantitative PCR(qRT-PCR)to verify the accuracy of the results.The results showed that the diame-ter and cross section of E8 muscle fiber in Rosecrown chickens and Cobb chickens were significantly smaller than those of E14(P<0.01),and the density of E8 muscle fiber was significantly higher than that of E14(P<0.01).Through bioinformatics analysis,2455 known miRNAs and 329 new miRNAs were identified in the four libraries,among which 499 differentially expressed miRNAs were screened in E8 and E14(C8T vs C14T_R8T vs R14T).GO enrichment results showed that differential miRNA tar-get genes were significantly enriched in functional items such as cell migration,metabolic process,cell proliferation,cell development and small molecule binding.KEGG enrichment analysis showed that a large number of target genes were enriched in signaling pathways related to cell turnover and metabolism,as well as signaling pathways related to muscle development such as TGF-β,Notch,Hedgehog,Wnt and FoxO.miRNA-mRNA joint analysis,it was found that miR-3594-3p-APOBEC2,miR-1635-FHL3/MEF2D.The results of miRNA-mRNA joint analysis showed that miR-193-3p-HDAC3,miR-1724-PRKAG3,miR-1698-PRKAG3 interaction pairs might be the key factors regulating chicken embryonic muscle fiber formation and muscle development.This study showed that the formation of muscle fiber in chicken embryonic period was regulated by a variety of miRNAs and signaling pathways.The miRNAs such as miR-3594-3p,miR-1635,miR-193-3p,miR-1724 and miR-1698 were screened out,which might play an important role in the formation of muscle fiber.