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

关键词:  血管内皮生长因子受体1  RNA干扰  慢病毒  U937细胞系

Key words:  vascular endothelial growth factor receptor 1  RNA interference  lentivirus  U937 cell line  

摘要:

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—j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—TimePCR、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—TimePCR检测显示U937细胞干扰组VEGFR-1mRNA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。

Abstract:

Objective To construct the shRNA expression vector targeting human vascular endothelial growth factor receptor 1 (VEGFR-1) 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 packaged 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 7-5.98% as compared to that of the control by Real-Time PCR. Wes tern b lot al so showed decreased expres si on of the VEGFR- 1 protei n in the RNA interference group . Conclusion The shRNA expression vector effectively inhibits the expression of VEGFR-1 gene in U937 cell line on mRNA and protein levels.