β-arrestin2抗基因RNA表达载体的构建及其基因沉默效应鉴定

Construction of expression vectors expressing beta arrestin 2 antigene RNAs and identification of their gene silencing effects in vitro

目的吗啡耐受与神经元μ阿片受体持续脱敏感化有关,而μ阿片受体调控蛋白β-arrestin2在其中起着关键性作用,因此β-arrestin2有可能成为抑制吗啡耐受状态的有效靶点。文中构建携带β-arrestin2抗基因RNA的表达载体pGPU6/GFP/agRNAs,并评价其用于基因沉默研究的可行性。方法根据β-arrestin2基因转录起始位点序列,从-14处开始至+13处结束,每隔一个碱基,依次合成9条21个碱基长度的抗基因RNA片段,并分别构建入质粒表达载体pGPU6/GFP/Neo。利用脂质体Lipofectamine介导的基因转染技术,分别将各个表达载体转染NG108-15细胞,于转染后第5天分别取转染不同抗基因RNA片段的细胞提取总RNA,应用realtime PCR和Western blot检测细胞β-arrestin2的表达,评价其基因沉默效应。结果筛选出1个表达具有基因沉默效应的抗基因RNA表达载体pGPU6/GFP/Arrb9。Realtime PCR结果显示NG108-15细胞转染该片段后,β-arrestin2 mRNA和蛋白表达水平均下降(P<0.05),下降程度达95%。结论携带β-arrestin2抗基因RNA的表达载体可有效下调NG108-15细胞β-arrestin2的表达,实现基因沉默效应。

Objective The development of morphine tolerance is correlated with the desensitization of the μ-opioid receptor in brain neurons.β-arrestin 2,a negative regulator of μ-opioid receptor,uncouples the receptors from G proteins and causes desensitization.This makes it possible for β-arrestin 2 to become an effective target for inhibiting morphine tolerance.The present study aimed to construct the expression vectors pGPU6/GFP/agRNAs containing β-arrestin 2 antigene RNAs and study their feasibility in gene silencing researches.Methods Nine 21-nucleotide agRNAs complementary to sequences throughout the-14 to ＋13 region of the β-arrestin 2 gene were synthesized and constructed into the expression vectors pGPU6/GFP/Neo,respectively.NG108-15 cells were transfected with pGPU6/GFP/agRNAs using Lipofectamine,and the expression of β-arrestin 2 in the NG108-15 cells was determined by real-time PCR and Western blot.The active pGPU6/GFP/agRNAs were defined as any agRNAs that yielded 50% inhibition of the β-arrestin 2 gene expression.Results pGPU6/GFP/Arrb9 was identified as the most potent inhibitor of the β-arrestin 2 gene expression.The transcription and expression of β-arrestin 2 in the NG108-15 cells was decreased by 95% after transfection（P0.05）.Conclusion The expression vector containing β-arrestin 2 antigene RNAs could effectively silence the gene expression in NG108-15 cells.