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Fam 229a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、FABP 4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Fam 229a,并对Fam 229a进行功能验证;最后利用PSIPRED软件、Phyre2.0软件和STRING网站预测Fam 229a下游功能及qPCR检测Fam 229a对早期成脂关键基因CEBPα、CEBPβ、ZFP 423和ZFP 521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FABP 4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Fam 229a之间存在结合作用,过表达miR-375-3p能显著抑制Fam 229a的表达(P<0.05),干扰后则表示相反结果。组织表达谱显示Fam 229a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,Fam 229a的表达量呈先上升后下降再上升趋势;过表达Fam 229a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Fam 229a后,结果相反;通过qRT-RCR技术和生物信息学预测了Fam 229a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Fam 229a抑制成脂分化,揭示了miR-375-3p靶向Fam 229a调控成脂分化的机制,丰富了miRNA的分子调控网络。
The study aimed to explore the regulatory effect of miR-375-3p on porcine adipocyte generation and its action mechanism targeting Fam 229a.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 Fam 229a as a downstream target of miR-375-3p,and the function of Fam 229a was subsequently validated.Finally,the downstream functions of Fam 229a 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,ZFP 423,and ZFP 521 regulated by Fam 229a.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,PPARγand FABP 4 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 Fam 229a was found through qRT-RCR,binding site prediction and dual luciferase assay.Overexpression of miR-375-3p could significantly inhibit the expression of Fam 229a(P<0.05).After interference,the opposite result is indicated.Tissue expression profile showed that Fam 229a was relatively high in liver and perirenal lipids.The temporal expression profile showed that the expression of Fam 229a increased first,then decreased and then increased during the differentiation of porcine primary precursor adipocytes.After overexpression of Fam 229a,the lipid drops were significantly decreased,and the mRNA levels of adipocyte differentiation marker genes CEBPαand PPARγwere significantly decreased(P<0.05),while the results were opposite after interfering with Fam 229a.The downstream mechanism of Fam 229a 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 Fam 229a inhibits adipogenic differentiation,revealing the regulation mechanism of adipogenic differentiation by miR-375-3p targeting Fam 229a,and enriching the molecular regulatory network of miRNA.
作者
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
YANG Yuting;CHEN Guoliang;CHANG Qiaoning;BAO Wu;LIU Jingchao;JI Mengting;RONG Xiaoyin;GUO Xiaohong;YANG Yang;LI Bugao(College of Animal Science,Shanxi Agricultural University,Taigu 030801,China)
出处
《畜牧兽医学报》
北大核心
2025年第3期1120-1133,共14页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金面上项目(32272846)
山西省重点研发计划项目(202102140601005)
山西省基础研究计划(202403021211043)。
作者简介
杨宇婷(1998-),女,山西祁县人,硕士,主要从事动物遗传育种与繁殖研究,E-mail:16635048175@163.com;通信作者:李步高,主要从事动物遗传育种与繁殖研究,E-mail:jinrenn@163.com;通信作者:杨阳,主要从事动物营养与饲料科学研究,E-mail:yangyangyh@163.com。
