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目的构建精氨酰-甘氨酰-天冬氨酸(RGD)介导的穿膜肽(TAT)-血管内皮细胞生长因子受体(KDR)siRNA融合基因慢病毒载体,探讨其对人肺癌A549细胞的抑制效果。方法设计合成编码RGD的2条寡核苷酸链,克隆到p GC/TAT-KDR siRNA载体的TAT下游,构建TAT-RGD-KDR siRNA融合基因载体;慢病毒包装并感染A549细胞;通过Western blot和real-time PCR检测A549细胞KDR基因表达水平;通过噻唑蓝法、双色法流式细胞仪和Transwell侵袭实验检测KDR基因沉默对A549细胞凋亡和侵袭力的影响。结果测序和Blast比对证实重组载体中的RGD序列与设计一致;TAT-RGD-KDR siRNA融合基因慢病毒载体转染A540细胞KDR mRNA和蛋白表达水平分别为(22.7±3.9)%和(19.3±2.7)%,明显低于TAT-KDR siRNA融合基因慢病毒载体转染组,差异有统计学意义(P<0.01);该载体具有较强的抑制A549细胞增殖、促进细胞凋亡和抑制细胞体外侵袭力的作用。结论 TAT-RGD-KDR siRNA融合基因慢病毒载体通过抑制细胞KDR mRNA和蛋白表达,抑制A549细胞增殖和侵袭、促进细胞凋亡。
Objective To construct a lentiviral vector containing arginyl - glycyl - aspartic acid (RGD) - mediated transmembrane peptide (TAT) - vascular endothelial growth factor receptor (KDR) siRNA fusion gene and investigate its effect on human lung cancer A549 Inhibitory effect of cells. METHODS: Two oligonucleotides encoding RGD were designed and synthesized. The downstream of TAT was cloned into p GC / TAT-KDR siRNA vector to construct TAT-RGD-KDR siRNA fusion gene vector. The lentivirus packaging and infecting A549 cells. The expression of KDR gene in A549 cells was detected by real-time PCR. The effect of KDR gene silencing on the apoptosis and invasiveness of A549 cells was detected by MTT assay, two-color flow cytometry and Transwell invasion assay. The results of sequencing and Blast comparison confirmed that the RGD sequence in the recombinant vector was consistent with the design. The KDR mRNA and protein expression of A549 cells transfected with TAT-RGD-KDR siRNA fusion lentiviral vector was (22.7 ± 3.9)% and (19.3 ± 2.7%), which was significantly lower than that of TAT-KDR siRNA fusion lentiviral vector transfected group, the difference was statistically significant (P <0.01); the vector had stronger inhibition of A549 cell proliferation, apoptosis and inhibition of cell in vitro The role of invasiveness. Conclusion TAT-RGD-KDR siRNA fusion lentiviral vector can inhibit the proliferation and invasion of A549 cells and promote cell apoptosis by inhibiting the expression of KDR mRNA and protein.