过表达miR-191抑制猪ADSCs向脂肪细胞分化

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（hormone sensitive 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 receptor γ）、C/EBPα（CCAAT/enhancer binding protein α）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.

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

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 during adipogenesis. 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 PPARγ, C/EBPα and A-FABP proteins gradually reduced for 8 days in ADSCs with miR-191 overexpression as compared to the control cells. 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.