IRS1、TCONS_00056481和miR-660在猪前体脂肪细胞分化过程中的时序表达及靶向关系鉴定

doi:10.11843/j.issn.0366-6964.2025.07.011

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (7): 3152-3163.doi: 10.11843/j.issn.0366-6964.2025.07.011

IRS1、TCONS_00056481和miR-660在猪前体脂肪细胞分化过程中的时序表达及靶向关系鉴定

王黎¹^,²(), 王秋妍¹(), 杨香香¹, 孙晓涵¹, 杜子孟¹, 尹丁¹, 张亚兰¹, 丁小玲¹, 殷宗俊¹, 郑先瑞¹, 张晓东¹, 丁月云¹^,*()

1. 安徽农业大学动物科技学院，安徽地方畜禽遗传资源保护与生物育种省级实验室，合肥 230036
2. 湖北省畜禽育种中心，武汉 430070

收稿日期:2024-12-10 出版日期:2025-07-23 发布日期:2025-07-25
通讯作者: 丁月云 E-mail:1934468966@qq.com;798750452@qq.com;dingyueyun@ahau.edu.cn
作者简介:王黎(1994-)，女，安徽巢湖人，硕士生，主要从事畜禽育种相关工作，E-mail: 1934468966@qq.com
王秋妍(2000-)，女，江苏南通人，硕士生，主要从事猪分子遗传育种方面的研究，E-mail: 798750452@qq.com
第一联系人：
王黎和王秋妍为同等贡献作者
基金资助:
国家重点研发计划项目(2021YFD1301200)；国家自然科学基金(31972531)；安徽省生猪产业技术体系项目(AHCYJSTX-05-03)

Temporal Expression of IRS1, miR-660 and lncRNA TCONS_00056481 During Porcine Preadipocyte Differentiation and Identification of Their Targeting Relationships

WANG Li¹^,²(), WANG Qiuyan¹(), YANG Xiangxiang¹, SUN Xiaohan¹, DU Zimeng¹, YIN Ding¹, ZHANG Yalan¹, DING Xiaoling¹, YIN Zongjun¹, ZHENG Xianrui¹, ZHANG Xiaodong¹, DING Yueyun¹^,*()

1. Anhui Provincial Laboratory of Animal Genetic Resources Protection and Biological Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China
2. Hubei Livestock and Poultry Breeding Center, Wuhan 430070, China

Received:2024-12-10 Online:2025-07-23 Published:2025-07-25
Contact: DING Yueyun E-mail:1934468966@qq.com;798750452@qq.com;dingyueyun@ahau.edu.cn

摘要/Abstract

摘要：

为探明IRS1、TCONS_00056481与miR-660之间的竞争性结合靶向关系。本研究分离培养猪皮下前体脂肪细胞，通过RT-qPCR检测TCONS_00056481、miR-660和IRS1在安庆六白猪(脂肪型)和大白猪(瘦肉型)背部脂肪中的表达水平及在猪前体脂肪细胞分化过程中的时序表达变化，Western blot检测IRS1蛋白在两猪种背脂中的表达情况，通过双荧光素酶报告基因检测系统鉴定miR-660与TCONS_00056481、IRS1之间的靶向结合关系。结果表明，IRS1、miR-660和TCONS_00056481在两猪种背脂中的表达水平均极显著差异(P＜0.01)，且在猪前体脂肪细胞分化过程中均显著差异性表达，说明三者在猪脂肪沉积和猪前体脂肪细胞分化的过程中具有潜在的调控作用；共转染TCONS_00056481野生型载体和miR-660 mimics的双荧光素酶活性极显著低于其对照组(P＜0.01)，共转染IRS1野生型载体和miR-660 mimics的双荧光素酶活性极显著低于其对照组(P＜0.01)；而共转染TCONS_00056481/IRS1突变型载体和miR-660 mimics的双荧光素酶活性与其对照组无显著差异(P＞0.05)，说明miR-660能够通过结合位点发挥对IRS1、TCONS_00056481的调控作用。研究初步验证了lncRNA TCONS_00056481可竞争性结合miR-660从而调控IRS1表达的靶向关系；IRS1、TCONS_00056481和miR-660可作为猪成脂调控的ceRNAs分子候选基因对进行后续研究。

关键词: IRS1, miR-660, TCONS_00056481, 双荧光素酶报告基因检测

Abstract:

The study aimed to explore the competitive binding and targeting relationship between TCONS_00056481, IRS1 and miR-660. The porcine subcutaneous preadipocytes were isolated and cultured, the expression levels of TCONS_00056481, miR-660 and IRS1 in the backfat tissues of Anqing Six-end-white pigs (fat type) and Large White pigs (lean type) and their temporal expression changes during porcine preadipocytes differentiation were detected by RT-qPCR, the expression levels of IRS1 protein in the backfat of the two pig breeds was detected by Western blot, and the targeted binding relationship between TCONS_00056481, IRS1and miR-660 were identified by dual luciferase reporter gene detection system in this study. The results showed that the expression levels of IRS1, miR-660 and TCONS_00056481 in the backfat of the two pig breeds were significantly different (P < 0.01), and the expression levels of IRS1, miR-660 and TCONS_00056481 were significantly different during the differentiation of porcine preadipocytes, indicating that IRS1, miR-660 and TCONS_00056481 had potential regulatory roles in pig fat deposition and porcine preadipocyte differentiation. The dual luciferase activity of co-transfected TCONS_00056481 wild-type vector and miR-660 mimics was significantly lower than that of its control group (P < 0.01), and the dual luciferase activity of co-transfected IRS1 wild-type vector and miR-660 mimics was significantly lower than that of its control group (P < 0.01); the dual luciferase activity of co-transfected TCONS_00056481/IRS1 mutant vector and miR-660 mimics was not significantly different from that of the control group (P>0.05), indicating that miR-660 could regulate IRS1 and TCONS_00056481 through binding sites. The study preliminarily verified that lncRNA TCONS_00056481 can competitively bind to miR-660 to regulate the expression of IRS1; IRS1, TCONS_00056481 and miR-660 can be used as candidate gene pairs of competing endogenous RNA molecules in regulating porcine lipid formation for further study.

Key words: IRS1, miR-660, TCONS_00056481, dual-luciferase reporter gene assay

中图分类号:

S828.2

王黎, 王秋妍, 杨香香, 孙晓涵, 杜子孟, 尹丁, 张亚兰, 丁小玲, 殷宗俊, 郑先瑞, 张晓东, 丁月云. IRS1、TCONS_00056481和miR-660在猪前体脂肪细胞分化过程中的时序表达及靶向关系鉴定[J]. 畜牧兽医学报, 2025, 56(7): 3152-3163.

WANG Li, WANG Qiuyan, YANG Xiangxiang, SUN Xiaohan, DU Zimeng, YIN Ding, ZHANG Yalan, DING Xiaoling, YIN Zongjun, ZHENG Xianrui, ZHANG Xiaodong, DING Yueyun. Temporal Expression of IRS1, miR-660 and lncRNA TCONS_00056481 During Porcine Preadipocyte Differentiation and Identification of Their Targeting Relationships[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3152-3163.

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引物名称 Primer name	引物序列 Primer sequence
TCONS-00056481	F: GACTGTCGCACCTGAAGAGC；R: TCCTCCTTGCCCTCCAAACA
IRS1	F: TGTAAAGAGTGGAGCAGAGGC；R: CTCAGCTCAACATCAGGGCT
miR-660	F: GGTACCCATTGCATATCGGAGTTG
GAPDH	F: GGAGAACGGGAAGCTTGTCA；R: GGTTCACGCCCATCACAAAC
U6	F: CTCGCTTCGGCAGCACA；R: AACGCTTCACGAATTTGCGT

组分Constituent	体积Volume
RNA	7 μL
Random Primer	1 μL
2×ES Reaction Mix	10 μL
EasyScript^Ⓡ RT/RI Enzyme Mix	1 μL
gDNA Remover	1 μL
25℃孵育10 min，42℃孵育15 min；85℃加热5 s灭活，－20℃保存

组分Constituent	体积Volume
miRNA	5 μL
2×miRNA RT Solution mix	10 μL
miRNA RT Enzyme mix	2 μL
RNase-free water	3 μL
混匀后37℃温水浴60 min；85℃加热5 min使酶失活，－20℃保存

组分Constituent	体积Volume
qPCR Mix	10 μL
Forward Primer(10 μmol·L^-1)	0.5 μL
Reverse Primer (10 μmol·L^-1)	0.5 μL
cDNA	2 μL
Water, nuclease-free	7 μL
Total volume	20 μL
94℃ 30 s；94℃ 5 s，40个循环；60℃ 30 s，40个循环

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IRS1、TCONS_00056481和miR-660在猪前体脂肪细胞分化过程中的时序表达及靶向关系鉴定

Temporal Expression of IRS1, miR-660 and lncRNA TCONS_00056481 During Porcine Preadipocyte Differentiation and Identification of Their Targeting Relationships

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