亲磷脂酸磷脂酶A1基因沉默降低MIN6细胞胰岛素分泌水平

Gene silencing of phosphatidic acid-preferring phospholipase A1 decreases insulin secretion in MIN6 cells

目的:探讨亲磷脂酸磷脂酶A1(PA-PLA1)基因沉默对小鼠分泌胰岛素细胞MIN6分泌功能的影响。方法:根据GenBank中小鼠PA-PLA1基因mRNA序列构建siRNA表达载体(pGPU6-PA-PLA1)并转染MIN6,实时荧光定量PCR和Western blot筛选有效干扰载体。将获得的有效干扰载体转染MIN6细胞48 h后进行葡萄糖刺激实验,检测其胰岛素分泌的变化。结果:酶切及测序证实成功构建了4个靶向PA-PLA1的siRNA表达载体。实时荧光定量PCR和Western blot分析显示siRNA表达载体pGPU6-PA-PLA1-1885具有较高的干扰效率,其转染的MIN6细胞PA-PLA1 mRNA水平降至对照组的46.3%,PA-PLA1蛋白水平降至对照组的33.9%,同时胰岛素分泌水平降至对照组的65.0%(P<0.05)。结论:PA-PLA1基因沉默可降低MIN6的胰岛素分泌水平。

Objective To explore the effect of the gene silencing of phosphatidic acid-preferringphospholipase A1 （PA-PLA1） on insulin secretion in mouse insulin-secreting cell line MIN6. Methods ThesiRNA expression vector of mouse PA-PLA1 gene targeting was constructed using mouse PA-PLA1 mRNAsequence available in GenBank, and MIN6 cells were transfected with the vector. Fluorescence quantitative PCRand Western-blotwere applied to screen efficient RNAi-vector. After transfection with obtained efficient RNAi-vectors for 48 hours, glucose-stimulated insulin secretion experiments were conducted, and the changes of insulinsecretion were examined. Results Four siRNA expression vectors of mouse PA-PLA1 gene targeting wereconfirmed to be successfully constructed by the analyses of enzyme cleavage and sequencing. The results offluorescence quantitative PCR and Western blot analyses indicated that the siRNA expression vectorpGPU6-PA-PLAl-1885was the most effective RNAi-vector in the four vectors. The expression levels of the PA-PLA1 mRNAand protein of the MIN6 cells transfectedwith pGPU6-PA-PLAl-1885 decreased to 46.3% and 33.9% of that ofthe control, respectively, and meanwhile the insulin secretion levels of the ceils decreased to 65.0% of that ofthe control （P 〈 0.05）. Conclusion The gene silencing of phosphatidic acid-preferring phospholipase A1 mightdecrease insulin secretion in MIN6 cells.