鸡circMICAL2的鉴定、组织表达谱分析及其功能预测

Identification of chicken circMICAL2,tissue expression profile analysis and its functional prediction

【目的】验证鸡环状RNA(circMICAL2)的真实性,研究circMICAL2在鸡肌肉生长发育中的潜在功能及调控机制。【方法】基于circMICAL2环状序列设计特异性引物采用PCR扩增完成其真实性验证,并测定RNase R和放线菌素D处理检测circMICAL2的内源稳定性。采集16日龄三黄鸡(雏鸡)和180日龄三黄鸡(成年鸡)的心脏、肝脏、肺、肾脏、肠、皮肤、胸肌和腿肌等8个组织样本,采用实时荧光定量PCR(qRT-PCR)分析circMICAL2在鸡不同发育时期的组织表达图谱;运用生物信息学对circMICAL2靶向miRNA和mRNA进行预测,并开展GO功能和KEGG通路富集分析。【结果】鸡circMICAL2真实存在,其环化稳定性强;circMICAL2在成年鸡和雏鸡各组织中广泛表达,且circMICAL2均在成年鸡和雏鸡的胸肌和腿肌肌肉中表达量最高,circMICAL2在成年鸡腿肌和胸肌中的表达量均显著高于雏鸡(P<0.05),circMICAL2与鸡肌肉生长发育调控密切相关。circMICAL2可靶向gga-miR-103-3p和gga-miR-130b-3p,调控下游225个潜在靶基因。circMICAL2的靶基因主要富集于TGF-β信号通路、MAPK信号通路、细胞周期等相关信号通路。【结论】鸡circMICAL2真实存在,circMICAL2的表达对鸡不同生长发育时期肌肉的生长具有重要调控作用。

【Objective】The purpose of this study is to verify the authenticity of chicken circular RNA(circMICAL2)and explore the function and regulation mechanism of circMICAL2 in chicken muscle growth and development.【Methods】Specific primers based on circMICAL2 circular sequences were designed by PCR amplification,and RNase R and actinomycin D treatments were performed to detect the endogenous stability of circMICAL2.Eight tissue samples from heart,liver,lung,kidney,intestine,skin,chest muscle and leg muscles of 16-day-old and 180-day-old chickens(adult chickens)were collected,and the tissue expression profile of circMICAL2 in different development periods was completed by real-time PCR(qRT-PCR);Bioinformatics was used to predict circMICAL2-targeted miRNA and mRNA,and to carry out GO function and KEGG pathway enrichment analysis.【Results】Chicken circMICAL2 really existed,and its cyclization stability was strong;circMICAL2 was widely expressed in all tissues of adults and chicks,and circMICAL2 was most highly expressed in their chest and leg muscles of adults and chicks,and the expression of circMICAL2 in both adult leg and thorax muscles was significantly higher than that in chicks(P<0.05),indicating that circMICAL2 was closely related to the regulation of chicken muscle growth and development.circMICAL2 targets gga-miR-103-3p and gga-miR-130b-3p regulated 225 downstream potential target genes.The target genes of circMICAL2 were mainly enriched in TGF-βsignaling pathway,MAPK signaling pathway,cell cycle and other related signaling pathways.【Conclusion】Chicken circMICAL2 really exists,and the expression of circMICAL2 plays an important role in regulating the growth of muscle during different growth and development periods.