Id2 shRNA慢病毒载体的构建及其转染后对胶质瘤细胞增殖和凋亡的影响

目的构建能高效敲减分化抑制因子2(Id2)基因的短发夹状小干扰RNA(shRNA)慢病毒载体,并观察敲减Id2对胶质瘤细胞株U251增殖和凋亡的影响。方法设计并合成特异性针对Id2基因的shRNA序列,将其构建到慢病毒载体pGCSIL-GFP中。以含有Id2基因的质粒为模板,扩增Id2基因cDNA序列的特异性片段,插入真核表达载体pEGFP-N1-3FLAG。构建含Id2基因质粒和不同靶点的RNAi慢病毒载体,共转染入工具细胞293T,筛选出适合浓度慢病毒感染U251细胞株。MTT法检测敲减Id2后胶质瘤细胞增殖的改变,RT-PCR检测Id2敲减后U251细胞caspase 3的表达。结果构建后载体的PCR鉴定及DNA测序结果与预期一致。与对照组相比,转染Id2 shRNA的U251细胞增殖降低,凋亡率升高,caspase3表达升高,差异有统计学意义(P<0.05)。结论成功构建的针对Id2基因的RNA干扰慢病毒载体体外转染可抑制胶质瘤细胞增殖并促进其凋亡。

Construction of lentiviral vector for RNA interference of Id2 gene and its effect on proliferation and apoptosis of gliomas cell line in vitro

ObjectiveTo construct a lentiviral vector carrying short hairpin RNA (shRNA) of human Id2 gene and to investigate its effect on the proliferation and apoptosis in glioma cell line U251.MethodsFour shRNA sequences targeting human Id2 gene were designed and synthesized. The shRNAs were inserted into the pGCSIL-GFP lentiviral vector. Id2 sequence was amplified from the plasmid which contained Id2 gene as verified by PCR, and was inserted into the pEGFP-N1-3FLAG vector. Lentiviral vector for RNAi of Id2 was constructed and was used to infect 293T cells. The infection efficiency was evaluated and then the lentiviral vector was used to infect U251 cell line. Western blotting analysis was employed to assess the gene silencing efficiency. Cell proliferation was tested by MTT. Cell apoptosis was observed by RT-PCR analysis of caspase 3 expression in U251 cells after infection.ResultsThe results of PCR analysis and DNA sequencing agreed to each other. Compared with the control group, the transfected U251 cells had significantly decreased cell proliferation, and increased apoptosis rate and caspase 3 expression (P<0.05).ConclusionWe have successfully constructed lentivirus RNAi vector of Id2, which can inhibit the proliferation and promote the apoptosis of glioma cells in vitro.