miR-375-3p靶向Fam229a调控猪前体脂肪细胞分化

doi:10.11843/j.issn.0366-6964.2025.03.014

摘要/Abstract

摘要：

旨在探究miR-375-3p对猪脂肪细胞生成的调控作用及靶向Fam229a的作用机制。本研究选取了饲养在相同条件下的10头30日龄的健康无病的公猪为研究对象，通过实时荧光定量PCR技术(qPCR)检测miR-375-3p在心脏、肝脏、脾脏等组织的表达分布及在脂肪细胞成脂诱导分化不同时期的表达水平；利用miRNA mimic和miRNA inhibitor过表达和干扰miR-375-3p，qPCR检测miR-375-3p对成脂关键基因CEBPα、PPARγ、FABP4表达的影响，油红O染色和统计分析检测成脂能力；通过生物信息学分析和双荧光素酶报告试验预测并鉴定miR-375-3p的下游靶标Fam229a，并对Fam229a进行功能验证；最后利用PSIPRED软件、Phyre2.0软件和STRING网站预测Fam229a下游功能及qPCR检测Fam229a对早期成脂关键基因CEBPα、CEBPβ、ZFP423和ZFP521表达的影响；上述试验方法均设立3个生物学重复。结果表明，miR-375-3p在猪各组织中广泛表达，在肺脏和肾周脂肪中相对表达量较高；时序表达谱显示，在猪原代前体脂肪细胞的分化过程中miR-375-3p的表达量呈先下降后上升趋势；过表达miR-375-3p后，猪原代前体脂肪细胞的脂滴明显增多，脂肪细胞分化标志基因CEBPα、PPARγ和FABP4的mRNA水平极显著上升(P < 0.01)，干扰miR-375-3p后，结果相反。为了探究miR-375-3p的作用机制，通过qRT-RCR、结合位点预测以及双荧光素酶试验发现了miR-375-3p与Fam229a之间存在结合作用，过表达miR-375-3p能显著抑制Fam229a的表达(P＜0.05)，干扰后则表示相反结果。组织表达谱显示Fam229a在肝脏和肾周脂肪中相对表达量较高；时序表达谱显示，在猪原代前体脂肪细胞的分化过程中，Fam229a的表达量呈先上升后下降再上升趋势；过表达Fam229a后，脂滴明显减少，脂肪细胞分化标志基因CEBPα、PPARγ的mRNA水平显著降低(P < 0.05)，干扰Fam229a后，结果相反；通过qRT-RCR技术和生物信息学预测了Fam229a调控成脂分化的下游机制。本研究结果表明，miR-375-3p能够促进猪原代前体脂肪细胞的成脂分化，其靶基因Fam229a抑制成脂分化，揭示了miR-375-3p靶向Fam229a调控成脂分化的机制，丰富了miRNA的分子调控网络。

关键词: 猪, 脂肪细胞分化, miR-375-3p, Fam229a

Abstract:

The study aimed to explore the regulatory effect of miR-375-3p on porcine adipocyte generation and its action mechanism targeting Fam229a. In this study, 10 healthy and disease-free boars of 30 days old were selected to be reared under the same conditions. Real-time fluorescence quantitative PCR (qPCR) was used to detect the expression distribution of miR-375-3p in the heart, liver, spleen and other tissues and the expression levels at different stages of adipogenic differentiation of adipocytes. Using miRNA mimics and miRNA inhibitors to overexpress and interfere with miR-375-3p, qPCR was employed to detect the effect of miR-375-3p on the expression of key adipogenic genes CEBPα, PPARγ, and FABP4. Oil red O staining and statistical analysis were used to assess the adipogenic capacity. Bioinformatics analysis and dual luciferase reporter assays were used to predict and identify Fam229a as a downstream target of miR-375-3p, and the function of Fam229a was subsequently validated. Finally, the downstream functions of Fam229a were predicted using PSIPRED software, Phyre2.0 software, and the STRING database, and qPCR was employed to detect the expression of early adipogenic key genes CEBPα, CEBPβ, ZFP423, and ZFP521 regulated by Fam229a. All of the above experimental methods were performed in triplicate to ensure biological reproducibility. The results showed that miR-375-3p was widely expressed in various tissues of pigs, with relatively high expression levels in lung and perirenal lipids. The temporal expression profile showed that the expression of miR-375-3p decreased first and then increased during the differentiation of porcine primary precursor adipocytes. After overexpression of miR-375-3p, the lipid droplets of porcine primary precursor adipocytes increased significantly, and the mRNA levels of adipocyte differentiation marker genes CEBPα, PPARγ and FABP4 increased significantly (P < 0.01). However, the results were reversed after interfering with miR-375-3p. In order to explore the action mechanism of miR-375-3p, a binding effect between miR-375-3p and Fam229a was found through qRT-RCR, binding site prediction and dual luciferase assay. Overexpression of miR-375-3p could significantly inhibit the expression of Fam229a (P < 0.05). After interference, the opposite result is indicated. Tissue expression profile showed that Fam229a was relatively high in liver and perirenal lipids. The temporal expression profile showed that the expression of Fam229a increased first, then decreased and then increased during the differentiation of porcine primary precursor adipocytes. After overexpression of Fam229a, the lipid drops were significantly decreased, and the mRNA levels of adipocyte differentiation marker genes CEBPα and PPARγ were significantly decreased (P < 0.05), while the results were opposite after interfering with Fam229a. The downstream mechanism of Fam229a regulating adipogenic differentiation was predicted by qRT-RCR technique and bioinformatics. This study results show that miR-375-3p can promote the adipogenic differentiation of porcine primary precursor adipocytes, and its target gene Fam229a inhibits adipogenic differentiation, revealing the regulation mechanism of adipogenic differentiation by miR-375-3p targeting Fam229a, and enriching the molecular regulatory network of miRNA.

Key words: pigs, adipocyte differentiation, miR-375-3p, Fam229a

中图分类号:

S828.2

杨宇婷, 陈国梁, 常巧宁, 鲍武, 刘靖超, 姬梦婷, 荣晓音, 郭晓红, 杨阳, 李步高. miR-375-3p靶向Fam229a调控猪前体脂肪细胞分化[J]. 畜牧兽医学报, 2025, 56(3): 1120-1133.

YANG Yuting, CHEN Guoliang, CHANG Qiaoning, BAO Wu, LIU Jingchao, JI Mengting, RONG Xiaoyin, GUO Xiaohong, YANG Yang, LI Bugao. miR-375-3p Targets Fam229a to Regulate Porcine Precursor Adipocyte Differentiation[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1120-1133.

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基因 Gene	引物序列(5′→3′) Primers sequence	产物长度/bp Products length	物种 Species
β-actin	F: GGCACCACACCTTCTACAATG R: GGGGTGTTGAAGGTCTCAAAC	158	鼠
CEBPα	F: GCTGAGCGACGAGTACAAGA R: GCTTGAACAAGTTCCGCAGG	191	鼠
CEBPβ	F: GCCAAGAAGACGGTGGACAA R: AGCTGCTCCACCTTCTTCTG	173	鼠
PPARγ	F: GGGGATGTCTCACAATGCCA R: CAGACTCTGGGTTCAGCTGG	107	鼠
FABP4	F: GATGCCTTTGTGGGAACCTG R: TCCTGTCGTCTGCGGTGATT	232	鼠
ZFP423	F: CAAGAAGTCCAAGGCTGAG R: TTGAGGTCGCACTGATTG	132	鼠
ZFP521	F: CTCAGCAGACCTCCGATAT R: GTAAGACCTCCAAGCAATACT	180	鼠
β-actin	F: CCAGGTCATCACCATCGG R: CCGTGTTGGCGTAGAGGT	158	猪
CEBPα	F: AGCCAAGAAGTCGGTAGA R: CGGTCATTGTCACTGGTC	150	猪
PPARγ	F: AGAGTATGCCAAGAACATCC R: AGGTCGCTGTCATCTAATTC	261	猪
FABP4	F: AAGTCAAGAGCACCATAACC R: GATACATTCCACCACCAACT	119	猪

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