shRNA沉默IRS-1基因对鼠前脂肪细胞分化和PPARγ表达的影响

The influence of shRNA silencing IRS-1 gene on the differentiation of mouse preadipocytes and PPARγ expression

目的观察胰岛素受体底物-1（IRS-1）基因表达受阻对小鼠3T3-L1前脂肪细胞分化和脂质过氧化物酶体增殖物激活受体γ（PPARγ）表达的影响。方法针对小鼠IRS-1基因开放阅读框上的2个区域合成短发夹核糖核酸[shRNA（M和MH）],以pGenesil-1vector为载体构建带绿色荧光蛋白（GFP）靶标的shRNA质粒。以脂质体LipofectaminTM2000介导转染3T3-L1前脂肪细胞,并设阴性对照（HK）载体转染组。细胞转染后,G418筛选稳定表达的阳性克隆并通过免疫印迹法鉴定。应用0.5mmol/L3-异丁基-1-甲基黄嘌呤（IBMX）、10-6mol/L地塞米松（DEX）和5μg/mL胰岛素（Ins）分别诱导小鼠3T3-L1前脂肪细胞空白对照组、HK载体转染组、M和MH转染组分化,在诱导分化的不同时间点收集细胞。用免疫印迹法检测PPARγ的表达。脂肪细胞内脂滴用油红O染色观察。结果与空白对照组、HK组和转染M组IRS-1shRNA质粒的3T3-L1前脂肪细胞相比,转染MH组IRS-1shRNA质粒的3T3-L1前脂肪细胞IRS-1蛋白表达水平降低70%以上;而MH转染组细胞PPARγ蛋白的表达与前3组3T3-L1前脂肪细胞比较无改变。前3组3T3-L1前脂肪细胞诱导分化为成熟脂肪细胞成功,PPARγ蛋白的表达在脂肪细胞分化成熟过程中逐渐增高。油红O染色显示,随着脂肪细胞的分化成熟,桔红色脂滴逐渐增多;而转染MH组IRS-1shRNA质粒的3T3-L1前脂肪细胞经诱导,油红O染色桔红色脂滴显着减少,直到分化的第8天,才见少许桔红色脂滴,且PPARγ蛋白的表达无变化。结论 IRS-1基因沉默后可抑制3T3-L1前脂肪细胞的分化,并在分化过程中抑制PPARγ的表达,说明IRS-1通过调节PPARγ的表达或与PPARγ共同作用在3T3-L1前脂肪细胞的分化中发挥决定性作用。

Objective To observe the influence of insulin receptor substrate-1 （ IRS-1） gene silencing on the differentiation of mouse 3T3-L1 preadipocytes and peroxisome proliferator-activated receptor γ （ PPARγ） expression. Methods Two short hairpin RNA （ shRNA M and shRNA MH） target to IRS-1 gene coding region were designed and cloned into pGenesil-1-shRNA vector with green fluorescence protein （ GFP） tag. The pGenesil-1-IRS-1-shRNA plasmids were stably transfected into 3T3-L1 preadipocytes by LipofectaminTM 2000,and negative control transfected group （ HK） was set. The positive clones were screened by G418 and identified through measuring IRS-1 protein by Western blot. 3-isobutyl-1-methylxanthine （ IBMX） （ 0. 5 mmol/L） ,dexamethasone （ DEX） （ 10 -6 mol/L） and insulin （ Ins） （ 5 μg/mL） were applied into inducing the differentiation of 3T3-L1 preadipocytes in control group,HK control group,M group and MH group. The expression of PPARγ was determined by Western blot. The lipid droplets of 3T3-L1 preadipocytes were observed by Oil-Red O staining. Results The expression of IRS-1 protein of 3T3-L1 preadipocytes transfected with IRS-1 shRNA plasmid in MH group significantly decaeased 70% compared with the control group,HK control group and M group. The expression of PPARγ had no change compared with other 3 groups. IRS-1 gene silencing did not affect the expression of PPARγ protein in uninduced 3T3-L1 preadipocytes,but decreased the expression of PPARγ protein during the in vitro induced differentiation of 3T3-L1 preadipocytes. The lipid droplets count was reduced by Oil-Red O staining. Conclusions The silencing of the IRS-1 gene inhibits the differentiation of 3T3-L1 preadipocytes,and suppresses the expression of PPARγ protein in the differentiation of 3T3-L1 preadipocytes. IRS-1 plays an important role by regulating the expression of PPARγ protein in the differentiation of 3T3-L1 preadipocytes.