构建shRNA慢病毒载体抑制U937细胞株VEGFR-1基因的表达

Inhibition of VEGFR-1 gene expression in U937 cell line using Lentivirus-mediated shRNA

目的构建针对血管内皮生长因子受体1(vascular endothelial growth factor receptor 1,VEGFR-1)基因的siRNA前体表达载体,鉴定其对U937细胞株VEGFR-1基因干扰效率。方法体外设计并合成针对VEGFR-1基因的慢病毒shRNA干扰载体,通过PCR及测序证实载体构建成功。将慢病毒颗粒以最适滴度转染人单核白血病细胞株U937,应用Real-Time PCR、Western印迹法检测抑制效率。结果构建的慢病毒载体的序列通过PCR、测序等鉴定,与设计序列相同。Real-Time PCR检测显示U937细胞干扰组VEGFR-1 mRNA表达率下降75.98%,Western印迹法显示U937细胞干扰组VEGFR-1蛋白表达量较空白载体对照组明显减少。结论构建的shRNA表达载体可在mRNA和蛋白水平上有效抑制U937细胞株VEGFR-1基因的表达。

Objective To construct the shRNA expression vector targeting human vascular en- dothelial growth factor receptor 1 （VEGFR-I） gene and to detect its silencing effects on human monocytic leukemia cell line U937. Methods Three pairs of target segments were synthesized and cloned into pRNAT-U6.2 Lentiviral vector respectively and the vectors were identified by PCR and DNA sequencing. Then the expression vectors were transferred into U937 cell line to produce pack- aged lentivirus. After being infected with the packaged lentivirus, the expression of VEGFR-1 gene of U937 cell line at mRNA and protein level was detected by Real-Time PCR and Western blotting. Results PCR and DNA sequencing showed that the target segments were cloned into pRNAT- U6.2 lentiviral vector respectively. The expression rate of VEGFR-1 gene in U937 cells in the RNA interference group had dropped by 75.98% as compared to that of the control by Real-Time PCR.Western blot also showed decreased expression of the VEGFR-1 protein in the RNA interference group. Conelusion The shRNA expression vector effectively inhibits the expression of VEGFR-1 gene in U937 cell line on mRNA and protein levels.