Gene silencing of phosphatidic acid-preferring phospholipase A1 decreases insulin secretion in MIN6 cells / 实用医学杂志

ABSTRACT

Objective To explore the effect of the gene silencing of phosphatidic acid-preferring phospholipase A1 (PA-PLA1) on insulin secretion in mouse insulin-secreting cell line MIN6. Methods The siRNA expression vector of mouse PA-PLA1 gene targeting was constructed using mouse PA-PLA1 mRNA sequence available in GenBank, and MIN6 cells were transfected with the vector. Fluorescence quantitative PCR and 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 insulin secretion were examined. Results Four siRNA expression vectors of mouse PA-PLA1 gene targeting were confirmed to be successfully constructed by the analyses of enzyme cleavage and sequencing. The results of fluorescence quantitative PCR and Western blot analyses indicated that the siRNA expression vectorpGPU6-PA-PLA1-1885was the most effective RNAi-vector in the four vectors. The expression levels of the PA-PLA1 mRNA and protein of the MIN6 cells transfectedwith pGPU6-PA-PLA1-1885 decreased to 46.3% and 33.9% of that of the control, respectively, and meanwhile the insulin secretion levels of the cells decreased to 65.0% of that of the control (P < 0.05). Conclusion The gene silencing of phosphatidic acid-preferring phospholipase A1 might decrease insulin secretion in MIN6 cells.