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

Abstract

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

CLC Number:

S828.2

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.

Figures/Tables 9

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

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