鸡circMICAL2的鉴定、组织表达谱分析及其功能预测

doi:10.6048/j.issn.1001-4330.2024.05.027

新疆农业科学 ›› 2024, Vol. 61 ›› Issue (5): 1284-1291.DOI: 10.6048/j.issn.1001-4330.2024.05.027

鸡circMICAL2的鉴定、组织表达谱分析及其功能预测

古丽帕日·艾克拜¹(), 沈雪梅², 喻世刚¹^,², 王钢²(), 杨雅玲¹, 刘武军¹()

1.新疆农业大学动物科学学院,乌鲁木齐 830052
2.乐山师范学院/川南地方品种鸡产业化四川省高等学校工程研究中心,四川乐山 614000

收稿日期:2023-09-28 出版日期:2024-05-20 发布日期:2024-07-09
通信作者: 刘武军(1966- ),女,河南鹿邑人,教授,博士,硕士生/博士生导师,研究方向为动物遗传育种与繁殖,(E-mail)lwj_ws@163.com;
王钢(1979-),男,重庆綦江人,教授,研究方向为动物疫病监测与防控、养殖业废弃物处理与利用,(E-mail)Lswanggang@163.com
作者简介:古丽帕日·艾克拜(1998-),女,新疆吐鲁番人,硕士研究生,研究方向为动物遗传育种与繁殖,(E-mail)1310784945@qq.com
基金资助:
四川省科技计划-重点研发项目(2022YFN0039);乐山师范学院科技计划项目(DGZZ202002);乐山师范学院科技计划项目(22HX00051)

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

Gulipari Aikebai¹(), SHEN Xuemei², YU Shigang¹^,², WANG Gang²(), YANG Yaling¹, LIU Wujun¹()

1. College of Animal Science/Xinjiang Agricultural University, Urumqi 830052, China
2. Engineering Research Center of Sichuan Province Higher School of Local Chicken Breeds Industrialization in Southern Sichuan/Leshan Normal University, Leshan Sichuan 614000,China

Received:2023-09-28 Published:2024-05-20 Online:2024-07-09
Correspondence author: LIU Wujun(1966-),female,from Luyi, Henan,professor,research direction:Animal genetics and breeding and reproduction,(E-mail)lwj_ws@163.com;
WANG Gang(1979-),male,from Qijiang, Chongqing,professor,research direction:Monitoring and prevention and control of animal diseases,Treatment and utilization of aquaculture waste,(E-mail)Lswanggang@163.com
Supported by:
Sichuan Science and Technology Program(2022YFN0039);Leshan Normal University Science and Technology Program(DGZZ202002);Leshan Normal University Science and Technology Program(22HX00051)

摘要/Abstract

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

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

中图分类号:

S831

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.

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.

图/表 10

表1 引物序列信息

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

图1 鸡circMICAL2引物设计模式注:红色箭头为反向引物,绿色箭头为正向引物;circMICAL2的R→F编码序列由MICAL2基因外显子7、8、9区域形成的

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

图2 circMICAL2 PCR扩增产物电泳检测注:M为Marker;符号▶◀为正向引物,◁▷为反向引物

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

图3 circMICAL2首尾链接序列测序峰注:A为高通量测序获得的鸡circMICAL2序列;B为目的片段胶回收测序序列;红色三角处为circMICAL2环化接头

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

图4 circRNA对RNase R消化的抗性变化注(Note):n=3,**P<0.01

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

图5 放线菌素D处理的鸡原代肌肉细胞中circMICAL2和线性MICAL2基因的表达丰度变化

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

图6 circMICAL2组织表达谱注:*表示差异显著(P<0.05);以心脏作为1进行相对表达量比较

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

图7 circMICAL2-miRNA-mRNA调控网络

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

图8 circMICAL2靶基因的GO

Fig.8 GO analysis of the circMICAL2 target gene

图9 circMICAL2靶基因KEGG

Fig.9 KEGG analysis of the circMICAL2 target gene

参考文献 31

[1]	李向阳, 张莉. 2021年中国禽肉市场回顾及 “十四五” 时期展望[J]. 农业展望, 2022, 18(1): 33-39.
	LI Xiangyang, ZHANG Li. Review on China’s poultry market in 2021 and its outlook for the 14th five-year plan period[J]. Agricultural Outlook, 2022, 18(1): 33-39.
[2]	邓小英, 刘圣林, 胡浩, 等. CircRNA翻译功能的研究进展及问题[J]. 生理科学进展, 2022, 53(3): 234-238.
	DENG Xiaoying, LIU Shenglin, HU Hao, et al. Research advances and problems on the translation functions of CircRNA[J]. Progress in Physiological Sciences, 2022, 53(3): 234-238.
[3]	Bahn J H, Zhang Q, Li F, et al. The landscape of microRNA, Piwi-interacting RNA, and circular RNA in human saliva[J]. Clinical Chemistry, 2015, 61(1): 221-230. DOI PMID
[4]	Rochow H, Franz A, Jung M, et al. Instability of circular RNAs in clinical tissue samples impairs their reliable expression analysis using RT-qPCR: from the myth of their advantage as biomarkers to reality[J]. Theranostics, 2020, 10(20): 9268-9279. DOI PMID
[5]	Xie M Y, Yu T, Jing X M, et al. Exosomal circSHKBP1 promotes gastric cancer progression via regulating the miR-582-3p/HUR/VEGF axis and suppressing HSP90 degradation[J]. Molecular Cancer, 2020, 19(1): 112. DOI PMID
[6]	Rybak-Wolf A, Stottmeister C, Gla?ar P, et al. Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically expressed[J]. Molecular Cell, 2015, 58(5): 870-885. DOI PMID
[7]	Peng W, Zhu S X, Chen J L, et al. Hsa_circRNA_33287 promotes the osteogenic differentiation of maxillary sinus membrane stem cells via miR-214-3p/Runx3[J]. Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie, 2019, 109: 1709-1717.
[8]	Cai B L, Ma M T, Zhou Z, et al. circPTPN4 regulates myogenesis via the miR-499-3p/NAMPT axis[J]. Journal of Animal Science and Biotechnology, 2022, 13(1): 2. DOI PMID
[9]	Yue B L, Yang H Y, Wu J Y, et al. circSVIL regulates bovine myoblast development by inhibiting STAT1 phosphorylation[J]. Science China Life Sciences, 2022, 65(2): 376-386.
[10]	Yan J Y, Yang Y L, Fan X H, et al. circRNAome profiling reveals circFgfr2 regulates myogenesis and muscle regeneration via a feedback loop[J]. Journal of Cachexia, Sarcopenia and Muscle, 2022, 13(1): 696-712.
[11]	欧阳宏佳. 环状RNA对鸡胚胎骨骼肌发育的影响[D]. 广州: 华南农业大学, 2017.
	OUYANG Hongjia. Effect of circular RNA on skeletal muscle development of chicken embryos[D]. Guangzhou: South China Agricultural University, 2017.
[12]	Zhao J, Zhao X Y, Shen X 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]	徐海冬, 冷奇颖, PATRICIA Adu-Asiama, 等. 环状RNA的特征及其在畜禽中的研究进展[J]. 生物技术通报, 2018, 34(11): 56-69. DOI
	XU Haidong, LENG Qiying, ADUASIAMA PATRICIA, et al. Circular RNAs: research progress and its significance in birds and livestock[J]. Biotechnology Bulletin, 2018, 34(11): 56-69. DOI
[14]	刘洪飞. 牛肌肉组织中品种特异性circQTL的鉴定和功能分析[D]. 杨凌: 西北农林科技大学, 2022.
	LIU Hongfei. Identification and functional analysis of breed-specific circQTL in bovine muscle tissue[D]. Yangling: Northwest A & F University, 2022.
[15]	付晓伟, 欧阳永灏, 洪乐, 等. 基于高通量测序技术的胰腺癌环状RNA差异表达谱分析[J]. 安徽医科大学学报, 2023, 58(1): 101-108.
	FU Xiaowei, OUYANG Yonghao, HONG Le, et al. Analysis of differential expression profile of circRNA in pancreatic cancer based on high-throughput sequencing technology[J]. Acta Universitatis Medicinalis Anhui, 2023, 58(1): 101-108.
[16]	贺花, 徐倩颖, 黄永震, 等. 环状RNA概述及其在动物肌肉发育中的研究进展[J]. 黑龙江畜牧兽医, 2020(3): 32-35.
	HE Hua, XU Qianying, HUANG Yongzhen, et al. Overview of circRNAs and its research progress in animal muscle development[J]. Heilongjiang Animal Science and Veterinary Medicine, 2020(3): 32-35.
[17]	Liu R L, Liu X X, Bai X J, et al. Identification and characterization of circRNA in longissimus dorsi of different breeds of cattle[J]. Frontiers in Genetics, 2020, 11: 565085.
[18]	Wei X F, Li H, Yang J M, et al. Circular RNA profiling reveals an abundant circLMO7 that regulates myoblasts differentiation and survival by sponging miR-378a-3p[J]. Cell Death & Disease, 2017, 8(10): e3153.
[19]	Liu Y, Chen Q, Bao J J, et al. Genome-wide analysis of circular RNAs reveals circCHRNG regulates sheep myoblast proliferation via miR-133/SRF and MEF2A axis[J]. International Journal of Molecular Sciences, 2022, 23(24): 16065.
[20]	Ouyang H J, Chen X L, Wang Z J, et al. Circular RNAs are abundant and dynamically expressed during embryonic muscle development in chickens[J]. DNA Research: an International Journal for Rapid Publication of Reports on Genes and Genomes, 2018, 25(1): 71-86. DOI PMID
[21]	王卫振. 静原鸡circACLY调控成肌细胞增殖、分化和凋亡的机制研究[D]. 银川: 宁夏大学, 2022.
	WANG Weizhen. Study on the Mechanism of circACLY Regulating the Proliferation, Differentiation and Apoptosis of Myoblasts in Jingyuan Chicken[D]. Yinchuan: Ningxia University, 2022.
[22]	余娇, 黎镇晖, 聂庆华, 等. 环状RNA circZBTB10的鉴定及其对鸡骨骼肌细胞增殖的影响[J]. 中国家禽, 2018, 40(24): 7-11.
	YU Jiao, LI Zhenhui, NIE Qinghua, et al. Identification of circZBTB10 and its function on chicken myoblast proliferation[J]. China Poultry, 2018, 40(24): 7-11.
[23]	孙晓峰, 张伟伟, 王阳, 等. MiR-103在牛骨骼肌卫星细胞中的分化调节作用[J]. 黑龙江畜牧兽医, 2015(15): 39-43, 294.
	SUN Xiaofeng, ZHANG Weiwei, WANG Yang, et al. The role of miR-103 in the differentiation and regulation on bovine skeletal muscle satellite cells[J]. Heilongjiang Animal Science and Veterinary Medicine, 2015(15): 39-43, 294.
[24]	Wang Y C, Yao X H, Ma M, et al. MiR-130b inhibits proliferation and promotes differentiation in myocytes via targeting Sp1[J]. Journal of Molecular Cell Biology, 2021, 13(6): 422-432.
[25]	Xue J, Xue J W, Zhang J, et al. MiR-130b-3p/301b-3p negatively regulated Rb1cc1 expression on myogenic differentiation of chicken primary myoblasts[J]. Biotechnology Letters, 2017, 39(11): 1611-1619. DOI PMID
[26]	张菊香, 张鹏, 陈晓萍. TGF-β/肌肉生长抑制素信号通路对骨骼肌作用的研究进展[J]. 航天医学与医学工程, 2011, 24(3): 224-228.
	ZHANG Juxiang, ZHANG Peng, CHEN Xiaoping. Research progress on roles of TGF-β/myostatin signaling pathway in skeletal muscle[J]. Space Medicine & Medical Engineering, 2011, 24(3): 224-228.
[27]	葛瑶. TCEA3通过ANXA1介导TGF-β通路进而影响小鼠成肌细胞分化[D]. 哈尔滨: 东北农业大学, 2019.
	GE Yao. TCEA3 Promotes Differentiation of C2C12 Cells Via An Annexin A1-mediated TGF-β Signaling Pathway[D]. Harbin: Northeast Agricultural University, 2019.
[28]	胡思敏. 牛肌肉卫星细胞中抑制MSTN表达后对脂肪代谢相关基因的影响[D]. 呼和浩特: 内蒙古大学, 2015.
	HU Simin. Effect of inhibiting MSTN expression in bovine muscle satellite cells on genes related to fat metabolism[D]. Hohhot: Inner Mongolia University, 2015.
[29]	冯阳. MIR-133b,miR-214和miR-495通过MAPK信号通路调节成肌细胞增殖和分化的研究[D]. 武汉: 华中农业大学, 2011.
	FENG Yang. The study of MIR-133b, miR-214 and miR-495 regulating myoblast proliferation and differentiation through MAPK signaling pathway[D]. Wuhan: Huazhong Agricultural University, 2011.
[30]	Zou L X, Zhong Y Q, Li X, et al. 3D-printed porous tantalum scaffold improves muscle attachment via integrin-β1-activated AKT/MAPK signaling pathway[J]. ACS Biomaterials Science & Engineering, 2023, 9(2): 889-899.
[31]	Wen L, Shumao L, Guihuan L, et al. Integrative Analyses of miRNA-mRNA Interactions Reveal let-7b, miR-128 and MAPK Pathway Involvement in Muscle Mass Loss in Sex-Linked Dwarf Chickens[J]. International Journal of Molecular Sciences, 2016, 17(3): 276. DOI PMID

编辑推荐 0

Metrics

阅读次数

全文

HTML			PDF

最新录用	在线预览	正式出版	最新录用	在线预览	正式出版
0	0	1	0	0	9

来源	本网站	其他网站

次数	7	3
比例	70%	30%

摘要

130

最新录用	在线预览	正式出版

0	0	130

来源	本网站	其他网站

次数	46	84
比例	35%	65%

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

在线阅读

PDF下载

可视化

摘要/Abstract

引用本文

使用本文

图/表 10

参考文献 31

相关文章 15

编辑推荐 0

Metrics

[1]	王超, 徐文修, 李鹏程, 郑苍松, 孙淼, 冯卫娜, 邵晶晶, 董合林. 棉花苗期生长发育对土壤速效钾水平的响应[J]. 新疆农业科学, 2024, 61(9): 2132-2139.
[2]	张承洁, 胡浩然, 段松江, 吴一帆, 张巨松. 氮肥与密度互作对海岛棉生长发育及产量和品质的影响[J]. 新疆农业科学, 2024, 61(8): 1821-1830.
[3]	马百幻, 赵强, 谢佳, 徐开玥, 任若飞, 宋兴虎. 生物药剂复配对棉花黄萎病防治及生长发育的影响[J]. 新疆农业科学, 2024, 61(7): 1748-1756.
[4]	赵云, 冯国郡, 古丽扎提·巴孜尔别克, 胡相伟, 苏比努尔·卡德尔, 李鹏兵, 邵疆, 刘杰. 钾肥用量对滴灌谷子生长发育及产量的影响[J]. 新疆农业科学, 2024, 61(6): 1378-1385.
[5]	王润琪, 贾永红, 王玉娇, 刘跃, 李丹丹, 董艳雪, 古力尼尕尔·吐尔洪, 张路路, 张金汕, 石书兵. 不同滴灌量对匀播冬小麦生长发育和产量的影响[J]. 新疆农业科学, 2024, 61(5): 1048-1056.
[6]	李雪瑞, 翟梦华, 徐新龙, 孙明辉, 张巨松. 无人机喷施不同浓度缩节胺对棉花生长发育的影响[J]. 新疆农业科学, 2024, 61(5): 1085-1093.
[7]	姚洋, 董伟, 李海英, 赵晓钰, 廖和荣, 吴盈萍, 王刚, 黄贵杰. 不同羽系拜城油鸡生长发育规律及生长曲线拟合分析[J]. 新疆农业科学, 2024, 61(5): 1259-1267.
[8]	侯献飞, 宋贤明, 李强, 顾元国, 苗昊翠, 曾幼玲, 郭美丽, 贾东海. 水氮耦合对膜下滴灌红花生长发育及产量的影响[J]. 新疆农业科学, 2024, 61(4): 791-803.
[9]	孙明辉, 叶尔兰·木合塔尔, 翟梦华, 李雪瑞, 徐新龙, 张巨松. 不同种植模式和品种对棉花光合物质生产及产量的影响[J]. 新疆农业科学, 2024, 61(3): 537-546.
[10]	陈传信, 张永强, 聂石辉, 孔德鹏, 赛力汗·赛, 徐其江, 雷钧杰. 生物质炭施用量对滴灌冬小麦生长发育和产量的影响[J]. 新疆农业科学, 2023, 60(9): 2146-2151.
[11]	宋冰梅, 姜岩, 陈鑫, 张宇, 程宛楠, 潘洪生. 新型转基因高产棉花萌发期和苗期耐盐性与耐碱性评价[J]. 新疆农业科学, 2023, 60(9): 2239-2247.
[12]	魏迎凤, 张全成, 查慧, 王小丽, 王俊刚. 二甲戊灵对龙葵苗期主要生长发育和生理指标的影响[J]. 新疆农业科学, 2023, 60(8): 2013-2021.
[13]	束佳敏, 郭元印, 卫丁一, 刘浩然, 依斯马依力, 黄磊, 何子涵, 姚刚, 戴小华. 野山杏总黄酮对LPS诱导鸡肝炎的影响[J]. 新疆农业科学, 2023, 60(8): 2066-2073.
[14]	李家辉, 赵晓钰, 李海英, 张俐华, 张杰, 魏彦, 周军, 赵全庄, 李宗福. 也迷离鸡生长发育规律及体重体尺的相关性分析[J]. 新疆农业科学, 2023, 60(5): 1281-1291.
[15]	杨梦琪, 南珊珊, 张晓羊, 王海亮, 李佳橙, 牛俊丽, 聂存喜, 张文举. 发酵棉籽粕膳食纤维对黄羽肉鸡生长性能、屠宰性能及肠道形态的影响[J]. 新疆农业科学, 2023, 60(4): 1011-1019.