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

摘要/Abstract

摘要：

旨在通过探究miR-376c-5p与PIK3CA的靶向关系，揭示miR-376c-5p对3T3-L1前体脂肪细胞分化的影响, 探索与脂肪分化相关的miR-376c-5p在脂肪生长分化过程中的作用及可能的调控机制。本课题组前期挑选了相同饲养条件下健康的6只3岁巴什拜羊(大尾型)和6只野生盘羊×巴什拜羊F2代绵羊(小尾型)收集其尾部脂肪组织，通过对尾部脂肪的测序数据分析结果，筛选出差异表达的miR-376c-5p。并构建miR-376c-5p mimic过表达载体进行转染，通过qPCR检测PIK3CA、成脂分化标志基因的表达情况；结合油红O染色和bodipy染色检测成脂能力。分析miR-376c-5p在不同物种中的保守性，利用生物信息学软件预测miR-376c-5p的靶基因PIK3CA及其之间的结合位点和双荧光素酶报告试验验证miR-376c-5p与PIK3CA之间的靶向关系。与mimic NC组相比，过表达miR-376c-5p后，qPCR结果显示分化成脂标志基因(PPARγ、ADIPOQ、ACACA、FASN)的mRNA表达水平均极显著下调(P＜0.001)，油红O和Bodipy结果均显示，过表达miR-376c-5p后，脂滴生成减少，说明过表达miR-376c-5p抑制了3T3-L1前体脂肪细胞的成脂分化。通过预测发现miR-376c-5p与PIK3CA 3′UTR区存在结合位点，双荧光素酶报告基因分析显示，过表达miR-376c-5p显著抑制了含有PIK3CA 3′UTR片段载体的荧光活性(P＜0.05);过表达miR376c-5p也会显著抑制候选靶基因PIK3CA mRNA的表达量(P＜0.05)。以上结果说明miR-376c-5p可能通过靶向PIK3CA抑制脂肪生成，研究结果为miRNA在分子水平上的研究提供了一定的理论依据。

关键词: miR-376c-5p, PIK3CA, 脂肪沉积

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

中图分类号:

S826.2

宋湘怡, 曹行, 刘武军, 蒋琳. miR-376c-5p靶向PIK3CA对前体脂肪细胞脂肪沉积的影响[J]. 畜牧兽医学报, 2025, 56(10): 4914-4924.

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.

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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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