过表达miR-191抑制猪ADSCs向脂肪细胞分化

Overexpression of MiR-191 Inhibits the Differentiation of Porcine ADSCs to Matured Adipocytes

在脂肪组织发育过程中存在许多差异表达的microRNAs(miRNAs),而miR-191是从实验室前期Solexa测序结果中筛选出的差异表达miRNAs之一.本研究对不同物种miR-191的成熟及前体序列进行同源性分析,并检测其在猪不同发育阶段各组织中的表达情况,发现miR-191的成熟及前体序列在不同物种间都非常保守,通过real-time qPCR检测发现,miR-191在猪脂肪组织不同发育阶段呈高丰度差异表达,其与Solexa测序结果一致.为了更好地研究miR-191在脂肪细胞分化过程中的功能,通过基因克隆的方法,利用Ad-Easy体系,构建过表达miR-191的腺病毒载体,转染293A细胞,成功包装出重组miR-191腺病毒并能高效侵染猪脂肪基质细胞(adipose derived stromal cells,ADSCs).通过real-time qPCR检测表明,感染重组miR-191腺病毒后的ADSCs能大量表达miR-191;油红O染色可以观察到,过表达miR-191的猪ADSCs在诱导分化后与对照组相比,脂滴明显减少.进一步研究发现,miR-191过表达的猪ADSCs中,SREBP-1C(sterol regulatory element binding protein-1C)和LPL(lipoprotein lipase)的mRNA水平显著降低,脂解关键基因HSL(hormonesensitive lipase)和ATGL(adipose triglyceride lipase)mRNA水平显著升高.同时,Western印迹结果显示,过表达miR-191的猪ADSCs在诱导分化过程中,PPARγ(peroxisome proliferator-activated receptorγ)、C/EBPα(CCAAT/enhancer binding proteinα)和A-FABP(adipocyte fatty acid binding protein)的蛋白表达水平显著下调.实验表明,过表达miR-191抑制了猪ADSCs的分化过程.这为深入研究miR-191在猪ADSCs分化过程中的调控机制奠定基础.

miRNAs are a class of endogenous small non-coding RNAs that play important roles in regulating gene expression. It has been demonstrated that numerous miRNAs were able to modulate adipocyte differentiation, including miR-191. The pre-miR-191 and matured miR-191 were conserved among mammals. The results of Solexa sequencing showed that the miR-191expression fluctuated duringadipogenesis. We examined the expression level of miR-191 in 3-day and 180-day old large white pigs, detected significant differences. To investigate the influence of miR-191 on adipocyte differentiation, an adenovirus vector was constructed with the Ad-Easy system. The adipose derived stromal cells （ADSCs） were infected by Ad-miR-191. The results indicated that overexpression of miR-191 dramatically reduced the mRNA expression level of SREBP-1C and LPL. Meanwhile, the lipolysis related genes HSL and ATGL mRNA expression levels increased remarkably. The overexpression of miR-191 also led to a notable reduction in lipid droplets in porcine pre-adipocytes as detected by oil red O staining. The Western blotting results indicated that the expression of PPART, C/EBPa and A-FABP proteins gradually reduced for 8 days in ADSCs with miR-191 overexpression as compared to the control ceils. In conclusion, we vertifed that miR-191 inhibited the ability of ADSCs differentiation in vitro, and demonstrated the importance of miR-191 in the biological mechanisms for adipogenesis regulation.