ShRNA靶向沉默VEGFR1基因对U937细胞增殖、迁移和凋亡的影响

Effect of VEGFR1 gene silencing by shRNA on proliferation,migration and apoptosis of U937 line

目的 探讨慢病毒载体介导shRNA（short harpin RNA,siRNA前体）靶向沉默血管内皮生长因子受体1（VEGFR-1）基因对U937细胞增殖、迁移和凋亡的影响.方法 构建针对VEGFR-1基因干扰序列和无关序列的shRNA慢病毒载体,与慢病毒包装质粒通过脂质体转染293T细胞,包装产生的病毒液感染U937细胞（U937-shVEGFR-1 KD组）.采用实时荧光定量PCR、Western blot法检测RNA干扰对U937细胞VEGFR-1 mRNA和蛋白表达的影响;ELISA法检测细胞培养上清液VEGF表达水平的变化;CCK-8法检测常规培养条件下和不同浓度阿糖胞苷（Ara-C）作用下细胞增殖率和抑制率变化;流式细胞术检测经Ara-C作用后细胞凋亡率变化;Transwell小室检测不同条件下细胞迁移数量的变化.结果 成功构建VEGFR-1基因shRNA慢病毒载体,转染U937细胞后,U937-shVEGFR-1 KD组细胞VEGFR-1 mRNA和蛋白表达均显著下降;细胞培养上清液VEGF表达水平明显下降,细胞增殖速度减慢;Ara-C作用下,U937-shVEGFR-1 KD组细胞增殖抑制率及凋亡率较U937无关序列shRNA阴性对照（NC）组和U937空白对照（CON）组细胞明显增高.无药物及VEGF作用下,U937-shVEGFR-1 KD组细胞迁移数量均低于U937 NC组和U937 CON组细胞;Bevacizumab作用下,U937 NC组和U937 CON组细胞迁移数量降低,而U937-shVEGFR-1 KD组细胞迁移数量无明显变化.结论 慢病毒载体介导的shRNA干扰VEGFR-1基因可有效抑制U937细胞增殖、迁移,并能够增强U937细胞对Ara-C的敏感性.

Objective To investigate the effects of lentivirus-mediated RNA interference targeting vascular endothelial growth factor receptor 1（VEGFR1）gene on the proliferation,migration and apoptosis of leukemic cell line U937.Methods Short hairpin RNAs（shRNA）targeting VEGFR-1 was synthesized and cloned into pRNAT-U6.2 lentiviral vector.The expression vectors were transfected into 293T cell line to produce packaged lentivirus.After infected with the packaged lentivirus,the expression of VEGFR-1 gene of U937 cells at mRNA and protein level was detected by real-time PCR and Western blot.VEGF production by the cells was determined by ELISA.Cell proliferation and survival under regular culture and in the presence of cytarabine（Ara-C）was determined by CCK-8 assay.Migration assays were performed by 5 μm pore transwell inserts.Results The lentiviral shRNA vector targeting VEGFR-1 was successfully constructed and transfected into U937 cells.The shRNA vector effectively inhibited the expression of VEGFR-1 gene in U937 cell line at mRNA and protein levels.As compared to that of the control,the proliferation rate of U937-sh-VEGFR-1 cells reduced;The VEGF production and migrated cell number of U937-shVEGFR-1 cells decreased dramatically.After treated with Ara-C,the inhibition rate and apoptotic rate of U937-shVEGFR-1cells increased significantly.The number of migrated cells in the KD group under regular culture and in the presence of VEGF was markedly lower than that in the NC group and CON group.Bevacizumab could decrease the number of migrated cells in the NC group and CON group,but could not in the KD group.Conclusions Lentivirus-mediated RNA interference targeting VEGFR1 gene reduces the proliferation,migrationof U937 cell line and enhances its sensitivity to Ara-C.