miR-27b及其靶基因PPARγ在肌内和皮下脂肪细胞中的差异表达分析

[目的] 探讨过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptor γ,PPARγ)在肌内和皮下脂肪细胞中的差异表达及其与脂质差异沉积的关系,并从miRNAs角度阐述PPARγ基因差异表达的机制。[方法] 用油红O染色法检测肌内前体脂肪细胞与皮下前体脂肪细胞的分化聚脂能力;采用qRT-PCR与Western blot检测PPARγ在肌内脂肪细胞和皮下脂肪细胞中的表达水平;通过生物信息学预测靶向PPARγ的miRNAs,利用qRT-PCR检测候选miRNAs在肌内脂肪细胞和皮下脂肪细胞中的表达水平;用双荧光素酶报告系统鉴定候选miRNAs与PPARγ的靶向性。[结果] 肌内前体脂肪细胞的分化聚脂能力显著高于皮下前体脂肪细胞(P<0.05);PPARγ的mRNA和蛋白在肌内脂肪细胞中的表达水平显著高于其在皮下脂肪细胞中的表达量(P<0.05);生物信息学预测得知miR-130a、miR-130b、miR-128、miR-301、miR-27a和miR-27b可以靶向PPARγ,其中miR-27a与miR-27b在肌内脂肪细胞中的表达量显著低于其在皮下脂肪细胞中的表达量(P<0.05);荧光素酶活性分析表明,miR-27b可以显著抑制PPARγ的荧光活性。[结论] 在体外,与皮下前体脂肪细胞相比,肌内前体脂肪细胞具有更强的分化能力。miR-27b在两种脂肪细胞中的差异表达可导致靶基因PPARγ的差异表达,进而引起肌内前体脂肪细胞与皮下前体脂肪细胞分化能力的差异。
[Objectives] The aim of this paper was to investigate the molecular mechanism underlying differential lipid deposition between porcine intramuscular and subcutaneous preadipocytes,peroxisome proliferator-activated recptor gamma(PPARγ)mRNA and protein levels were compared in these two types of adipocytes,the microRNAs(miRNAs)potentially targeting PPARγ gene were also predicted and verified. [Methods] The differentiation abilities of intramuscular and subcutaneous preadipocytes were assessed via oil red O staining. The mRNA and protein levels of PPARγ were detected by quantitative real-time reverse transcription-polymerase chain reaction(qRT-PCR)and Western blot. The potential miRNAs targeting PPARγ were predicted by bioinformatics prediction,and miRNAs expression levels were detected by qRT-PCR. The target relationship between the candidate miRNAs and PPARγ was analyzed by dual-luciferase reporter assay. [Results] Intramuscular preadipocytes had stronger differentiation ability(P<0.05). Both mRNA and protein levels of PPARγ were higher in intramuscular adipocytes(P<0.05). Six miRNAs,miR-130a,miR-130b,miR-128,miR-301,miR-27a and miR-27b,were predicted to potentially target PPARγ. Among the six miRNAs,miR-27a and miR-27b had significantly lower expression levels in intramuscular adipocytes(P<0.05). However,dual luciferase activity analysis revealed that only miR-27b could suppress PPARγ expression(P<0.05). [Conclusions] Intramuscular preadipocytes possess stronger differentiation ability than subcutaneous preadipocytes in vitro. We infer that the variance of miR-27b expression results in the diverse levels of PPARγ,and could be the reason for differential fat deposition between intramuscular and subcutaneous adipocytes in pigs