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

doi:10.6048/j.issn.1001-4330.2024.05.027

Abstract

Abstract:

【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-β signaling 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.

Key words: circRNA; muscle; chicken; tissue expression; growth and development

摘要：

【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。

关键词: 环状RNA, 肌肉, 鸡, 组织表达, 生长发育

CLC Number:

S831

Gulipari Aikebai, SHEN Xuemei, YU Shigang, WANG Gang, YANG Yaling, LIU Wujun. Identification of chicken circMICAL2, tissue expression profile analysis and its functional prediction[J]. Xinjiang Agricultural Sciences, 2024, 61(5): 1284-1291.

古丽帕日·艾克拜, 沈雪梅, 喻世刚, 王钢, 杨雅玲, 刘武军. 鸡circMICAL2的鉴定、组织表达谱分析及其功能预测[J]. 新疆农业科学, 2024, 61(5): 1284-1291.

Figures/Tables 10

Tab.1 primer sequence information

基因名称 Gene name	引物序列 Primer sequences 5'→3'	产物长度 Product length (bp)
正向引物 Divergent primer	F:GCAATTTGCTGTCCTACGCTC R:CCATTGGCCAGAAAGGTCAA	224
反向引物 Convergent primer	F:GCACTGGTGAGAGAAAGGCA R:CGTAGGACAGCAAATTGCCC	348
MICAL2	F:CTAAAGAGAGGGAGGGCAGGTC R:CCCTTCTCCAAAAGCATCCAG	192
GAPDH	F:GAGAAATTGTGCGTGACATCA R:CCTGAACCTCTCATTGCCA	152

Fig.1 The primer design strategy for PCR of chicken circMICAL2 Note: Red arrow indicates convergent primer, Green arrow indicates divergent primer;The R→F coding sequence of circMICAL2 is formed by the region of exons 7,8, and 9 of the MICAL2 gene

Fig.2 The circMICAL2 PCR amplification products were detected by electrophoresis Note: M indicates DNA Marker, the symbol▶◀indicates the divergent primer, and◁▷indicates the convergent primer

Fig.3 circMICAL2 Peak map of head-end linked sequence Note: A shows the chicken circMICAL2 sequence obtained from high-throughput sequencing;B Sequenced sequence after recovery of agarose gel;The circMICAL2 cyclization joint is shown in the red triangle

Fig.4 Changes of circRNA analysis of resistance to RNase R digestion

Fig.5 Changes in expression levels of circMICAL2 and linear MICAL2 genes in chicken primary muscle cells treated with DActinomycin D

Fig.6 circMICAL2 tissue expression profile Note:* indicates significant difference(P <0.05);The relative expression levels were compared with the heart as 1

Fig.7 Regulatory network diagram of circMICAL2-miRNA-mRNA

Fig.8 GO analysis of the circMICAL2 target gene

Fig.9 KEGG analysis of the circMICAL2 target gene

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