PTEN基因干扰对牛脂肪细胞凋亡的影响

Effect of PTEN Gene Interference on Apoptosis in Adipocytes of Cattle

实验旨在研究RNA干扰PTEN基因对牛脂肪细胞中p-Akt蛋白及凋亡蛋白Caspase-3表达的影响。将人工合成的靶向PTEN基因的3条干扰质粒Si-952组、Si-1331组、Si-1435组及阴性对照组(只加转染阴性对照质粒)和空白组(只加PBS缓冲液),在脂质体介导下转染牛脂肪细胞,用RT-qPCR方法检测并筛选干扰效率最高的1条Si RNA。Western blot法检测PTEN基因沉默后对p-Akt和Caspase-3表达的影响。结果表明:PTEN-Si-1331组干扰效率最高,转染48 h后抑制率可达85%;牛脂肪细胞转染Si-1331组Caspase-3蛋白表达水平极显著低于阴性对照组和空白组(P<0.01),而p-Akt蛋白表达水平显著高于阴性对照组和空白组(P<0.01)。上述结果表明,干扰PTEN基因能显著抑制Caspase-3的表达,提高p-Akt的表达,揭示低表达PTEN通过调控Akt信号通路抑制牛脂肪细胞凋亡。

The purpose of the study was to investigate the influence of PTEN gene silencing by RNA interference on the expressions of phospho-Akt and Caspase-3 in adipocytes of cattle.We transfected three chemically synthesized PTENSiRNA-952,PTEN-SiRNA-1331,PTEN-SiRNA-1435,negative control and blank control groups into adipocytes of cattle,and detected the interference efficiency with RT-qPCR in order to select the SiRNA with the highest interference efficiency.Then,the effect of the PTEN silencing on the activation of p-Akt and Caspase-3 was detected by Western blot analysis.The result showed that PTEN-SiRNA-1331 had the highest interference efficiency,and its interference efficiency was approximately 85%at 48h after the transfection.The expression of PTEN protein and Caspase-3 protein in shRNA plasmid transfection group was significantly lower than those in negative control and blank control groups(P<0.01).The expression of p-Akt protein in shRNA plasmid transfection group was significantly higher than those in negative control and blank control groups(P<0.01).We conclude that down regulation of PTEN by RNAi significantly inhibits the expression of Caspase-3 and increase the expression of p-Akt,indicating that PTEN gene showed a certain inhibitory effect on apoptosis of adipocytes by Akt pathway.