miR-376c-5p靶向PIK3CA对前体脂肪细胞脂肪沉积的影响

doi:10.11843/j.issn.0366-6964.2025.10.013

Abstract

Abstract:

This study aimed to investigate the targeting relationship between miR-376c-5p and PIK3CA to elucidate how miR-376c-5p influences 3T3-L1 preadipocyte differentiation, exploring its role and regulatory mechanisms in adipogenesis. In the early stage, the research group selected 6 healthy 3-year-old Bashbay sheep (large-tailed type) and 6 F2 generation sheep of wild Argali×Bashbay sheep (small-tailed type) under the same feeding conditions, their tail adipose tissues were collected, and screened out the differentially expressed miR-376c-5p through the sequencing data analysis results of the tail fat. Then, an miR-376c-5p mimic overexpression vector was constructed for transfection, and the expression levels of PIK3CA and adipogenic differentiation marker genes were detected by qPCR. The adipogenic capacity was detected by combining Oil Red O staining and Bodipy staining. The conservation of miR-376c-5p in different species was analyzed, and bioinformatics software was used to predict the target gene(PIK3CA) of miR-376c-5p and the binding sites between them. Additionally, dual-luciferase reporter assay was performed to verify the targeting relationship between miR-376c-5p and PIK3CA. Compared with the mimic NC group, qPCR results showed that overexpression of miR-376c-5p significantly downregulated the mRNA expression levels of key adipogenic differentiation marker genes (PPARγ, ADIPOQ, ACACA and FASN)(P < 0.001). Both Oil Red O and Bodipy staining results demonstrated a reduction in lipid droplet formation following miR-376c-5p overexpression, indicating that overexpression of miR-376c-5p inhibited adipogenic differentiation in 3T3-L1 preadipocytes. Bioinformatic prediction revealed a potential binding site between miR-376c-5p and the 3′UTR of PIK3CA. Dual-luciferase reporter assay further confirmed that overexpression of miR-376c-5p significantly suppressed the luciferase activity of a vector containing the PIK3CA 3′UTR fragment (P < 0.05). Moreover, overexpression of miR-376c-5p markedly reduced the mRNA expression level of the candidate target gene PIK3CA (P < 0.05). These findings suggest that miR-376c-5p may inhibit adipogenesis by targeting PIK3CA, providing a theoretical basis for further exploration of miRNA functions at the molecular level.

Key words: miR-376c-5p, PIK3CA, fat deposition

CLC Number:

S826.2

SONG Xiangyi, CAO Hang, LIU Wujun, JIANG Lin. miR-376c-5p Regulates Lipid Accumulation in Preadipocytes by Targeting PIK3CA[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 4914-4924.

Figures/Tables 7

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基因Gene	序列(5′→3′)Sequence
miR-376c-5p	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAACAT
	F：CGCGGTGGATATTCCGTCT
	R：AGTGCAGGGTCCGAGGTATT
PIK3CA	F：CACCTGAACAGACAAGTAGAGGC
	R：GCAAAGCATCCATGAAGTCTGGC
PPARγ	F：CGGAAGCCCTTTGGTGACTT
	R：CCTCGATGGGCTTCACGTTC
ACACA	F：CCGCCAGCCTGAGTTCTTTT
	R：ATCGGGAGTGCTGGTTTAGC
FASN	F：GGGTCTATGCCACGATTC
	R：GTGTCCCATGTTGGATTTG
ADIPOQ	F：GGCAGGAAAGGAGAACCTGG
	R：AGCCTTGTCCTTCTTGAAGAG
U6	F：TCGCTTCGGCAGCACATA
	R：TTTGCGTGTCATCCTTGC
18S	F：GTAACCCGTTGAACCCCATT
	R：CCATCCAATCGGTAGTAGCG

物种 Species	名称 Name	登录号 Accession number	引物长度/nt Length	序列(5′→3′) Sequence
绵羊Sheep	oar-miR-376c-5p	MIMAT0019279	21	GUGGAUAUUCCGUCUAUGUUU
小鼠Mouse	mmu-miR-376c-5p	MIMAT0005295	22	GUGGAUAUUCCUUCUAUGUUUA
大鼠Rat	rno-miR-376c-5p	MIMAT0017219	21	GUGGAUAUUCCUUCUAUGUUU
人Human	hsa-miR-376c-5p	MIMAT0022861	21	GGUGGAUAUUCCUUCUAUGUU
猕猴Macaca mulatta	mml-miR-376c-5p	MIMAT0026863	22	GUGGAUAUUCCUUCUAUGUUUA

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miR-376c-5p Regulates Lipid Accumulation in Preadipocytes by Targeting PIK3CA

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