miR-301b对山羊肌内脂肪细胞分化的调控作用

Regulation of miR-301b on Goat Intramuscular Adipocyte Differentiation

旨在明确miR-301b对山羊肌内脂肪细胞分化的调控作用,探讨其发挥调控作用的可能机制。利用化学合成的miR-301b mimics和miR-301b siRNA,通过脂质体转染法在山羊肌内脂肪细胞过表达和干扰miR-301b,并通过油红O染色、qPCR和生物信息学分析等方法,从细胞形态学和mRNA水平明确miR-301b对山羊肌内脂肪细胞分化的调控作用,并初步探究其可能的作用机制。结果表明,过表达miR-301b效率约29290倍,细胞形态学结果显示,过表达miR-301b后山羊肌内脂肪细胞脂滴积聚减少,甘油三酯合成降低。qPCR结果显示,过表达miR-301b后成脂标志基因CEBPα、PPARγ、SREBP1、LPL表达水平极显著下调(P<0.01)。miR-301b干扰效率约60%,干扰miR-301b表达后,细胞形态学结果显示,山羊肌内脂肪细胞脂滴积聚增加,甘油三酯合成升高。qPCR结果显示,干扰miR-301b后成脂标志基因AP2、CEBPα、CEBPβ、PPARγ、SREBP1、LPL表达水平极显著上调(P<0.01)。通过对miR-301b生物信息学分析,推测KLF3为miR-301b的靶标基因,qPCR验证发现过表达miR-301b后显著抑制KLF3的mRNA表达水平(P<0.05),干扰miR-301b后显著促进KLF3的表达水平(P<0.05)。过表达miR-301b抑制山羊肌内前体脂肪细胞分化,同时KLF3表达水平下调;干扰miR-301b促进山羊肌内前体脂肪细胞分化,KLF3表达水平上调,推测miR-301b可能通过KLF3抑制山羊肌内脂肪细胞分化。

The purpose of this experiment is to clarify the regulatory effect of miR-301b on goat intramuscular adipocyte differentiation and to explore the possible mechanism of its regulatory effect.Chemically-synthesized miR-301b mimics and miR-301b siRNA were used to overexpress and interfere miR-301b in goat intramuscular adipocytes by liposome transfection,and the regulatory effect of miR-301b on goat intramuscular adipocyte differentiation was clarified from the cell morphology and mRNA level by oil red O staining,qPCR and bioinformatics analysis,and its possible mechanism was explored preliminary.The results showed that the overexpression efficiency of miR-301b was about 29290 times.The results of cell morphology showed that the accumulation of lipid droplets and triglyceride synthesis of goat intramuscular adipocytes decreased after the overexpression of miR-301b.qPCR results showed that expressions of lipogenic marker gene CEBPα,PPARγ,SREBP1 and LPL in the overexpressed miR-301b significantly reduced(P<0.01).The interference efficiency of miR-301b was about 60%.After the expression of miR-301b was interfered,the results of cell morphology showed that the accumulation of lipid droplets in goat intramuscular adipocytes increased and the synthesis of triglycerides increased.qPCR results showed that interfering miR-301b significantly promoted expressions of adipogenic marker gene AP2,CEBPα,CEBPβ,PPARγ,SREBP1 and LPL.Through bioinformatics analysis of miR-301b,it was speculated that KLF3 was the target gene of miR-301b.qPCR verification found that overexpression of miR-301b significantly inhibited the mRNA expression of KLF3,and interference to miR-301b significantly promoted the expression of KLF3.In conclusion,overexpression of miR-301b inhibited the differentiation of intramuscular preadipocytes in goats,and down-regulated the expression of KLF3;interfering miR-301b promoted the differentiation of intramuscular preadipocytes in goats,and the expression of KLF3 was up-regulated.Thus it is speculated that miR-301b may inhibit the differentiation of goat intramuscular adipocytes through KLF3.