Sirt3基因RNA干扰慢病毒载体及稳定表达的人神经母细胞瘤SH-SY5Y细胞株的构建

RNA interference of Sirt3 in human neuroblastoma SH-SY5Y cell

目的构建Sirt3基因RNA干扰慢病毒载体,建立Sirt3基因稳定干扰的人神经母细胞瘤细胞SHSY5Y细胞株。方法从Genbank中检索到Sirt3基因序列,根据此序列设计Sirt3基因的4条siRNA干序列和1条阴性对照序列,将它们分别与含绿色荧光蛋白(GFP)编码基因的线性化慢病毒载体连接,获得4种重组慢病毒质粒。将4种重组的干扰病毒质粒分别与慢病毒包装质粒共转染293T细胞,测定病毒滴度。将4种构建的慢病毒载体感染SH-SY5Y细胞,采用Real-time PCR和Western blot检测Sirt3的沉默效果,筛选出最有效干扰Sirt3基因表达组。结果测序证实成功构建了4种Sirt3基因RNA干扰慢病毒载体,病毒滴度分别为:LV-SIRT3-RNAi-18×10^8 TU/mL、LV-SIRT3-RNAi-23×10^8 TU/mL、LV-SIRT3-RNAi-38×10^8 TU/mL、LV-SIRT3-RNAi-48×10^8 TU/mL。与空白对照组(CON)和阴性对照组(NC)相比,LV-Sirt3-RNAi-3组mRNA表达水平及蛋白表达水平显著下降(P<0.001,P<0.001)。结论成功构建Sirt3基因RNA干扰慢病毒载体,并筛选出最佳干扰序列组,获得稳定沉默Sirt3表达的SH-SY5Y细胞株,为后续研究Sirt3在帕金森病细胞模型中的作用奠定了基础。

Objective To construct a lentiviral vector for RNA interference of the Sirt3 gene and to establish a Sirt3 knockdown human neuroblastoma SH-SY5Y cell line.Methods According to the Sirt3 nucleotide sequence archived in the GenBank database,four siRNAs targeting Sirt3 and one negative control were designed,and cloned into a linear vector containing the green fluorescent protein(GFP)gene to produce four recombinant lentivirus plasmids.The recombinant plasmids and helper plasmids were transfected into 293T cells,and the titer of the virus was determined.SHSY5Y cells were infected with the constructed lentivirus,and the silencing effect on Sirt3 was accessed by real-time PCR and Western blot.The lentivirus-infected cells were screened for the most significant Sirt3 knockdown.Results Recombinant lentiviral vectors expressing siRNAs targeting Sirt3 were successfully constructed and confirmed by DNA sequencing.The viral titers of the recombinant lentivirus were as follows:LV-SIRT3-RNAi-18×10^8 TU/mL,LV-SIRT3-RNAi-23×10^8 TU/mL,LV-SIRT3-RNAi-38×10^8 TU/mL,and LV-SIRT3-RNAi-48×10^8 TU/mL.The levels of Sirt3 mRNA and SIRT3 protein in the LV-Sirt3-RNAi-3 group were significantly less than those in the negative control group and the blank control group(P<0.001,P<0.001).Conclusions A lentivirus vector for RNA interference of Sirt3 was successfully constructed and SH-SY5Y cell lines with Sirt3 gene knockdown were selected,which will be useful for future research of Sirt3 function in Parkinson’s disease cell models.