Brahma相关基因-1调控乏氧诱导因子-1α参与结直肠癌血管生成

Effect of Brahma-related gene-1 on angiogenesis of colorectal cancer through hypoxia inducible factor-1α

目的观察交配型转换/蔗糖不发酵(SWI/SNF)复合体调节亚基Brahma相关基因-1(Brg-1)对结直肠癌血管生成的影响。方法对结肠癌细胞系LoVo细胞感染sh-Brg-1慢病毒(Lenti-sh-Brg-1组)和对照病毒(Lenti-con组),通过蛋白质印迹法(Western blot)及实时定量聚合酶链反应(Real-time PCR)检测常氧及乏氧状态下乏氧诱导因子-1α(HIF-1α)、血管内皮生长因子-A(VEGF-A)的蛋白和mRNA水平的变化,运用体外微管形成实验观察低表达Brg-1对结直肠癌血管形成的影响,组间比较采用t检验。结果Western blot结果证明,与Lenti-con组比较,Lenti-sh-Brg-1组细胞中Brg-1蛋白表达量明显降低。在常氧及乏氧状态下低表达Brg-1可以明显降低HIF-1α及VEGF-A在蛋白表达量。在常氧状态下,Lenti-sh-Brg-1组细胞Brg-1(con 1.00±0比sh-Brg-10.53±0.08,t=5.292,P<0.01),HIF-1α(con 1.00±0比sh-Brg-10.61±0.07,t=5.380,P<0.01),VEGF-A mRNA水平低于Lenti-con组(con 1.00±0比sh-Brg-10.38±0.10,t=6.169,P<0.01);在乏氧状态下,Lenti-sh-Brg-1组细胞Brg-1(con 1.00±0比sh-Brg-10.07±0.02,t=44.680,P<0.01),HIF-1α(con 1.00±0比sh-Brg-10.21±0.05,t=14.280,P<0.01),VEGF-A mRNA水平低于Lenti-con组(con 1.00±0比sh-Brg-10.15±0.06,t=13.960,P<0.01)。最后证实在常氧和乏氧条件下,Brg-1低表达细胞条件培养基组的人脐静脉内皮细胞体外成管数目低于对照组(常氧,con 55.67±7.22比sh-Brg-122.33±5.36,t=3.706,P<0.05;乏氧,con 66.33±7.62比sh-Brg-135.68±4.91,t=3.382,P<0.05)。结论在LoVo细胞系中低表达Brg-1可以通过下调HIF-1α抑制血管形成。

Objective To investigate the effect of regulatory subunit Brahma-related gene-1(Brg-1)of Switch/Sucrose non-fementable(SWI/SNF)complex on hypoxia inducible factor-1α(HIF-1α)and angiogenesis of colorectal cancer LoVo cell line.Methods LoVo cell line was transfection with lenti-sh-Brg-1.The efficiency of transfection was verified by Western blotting.The protein level and mRNA relative level of HIF-1αand vascular endothelia growth factor-A(VEGF-A)were measured by western blotting and real-time quantitative polymerase chain reaction(Real-time PCR).Tube formation of human umbilical vein endothelial cells(HUVEC)in vitro was employed to probe the influence of Brg-1 on colon cancer angiogenesis.The independent sample t test was used for the comparison between two groups.Results The stable cell line was certified with low expression Brg-1.Knock-down of Brg-1 generated reduced the expression of both HIF-1αand VEGF-A on protein level.In Lenti-sh-Brg-1 group,the expression of Brg-1(con 1.00±0 vs.sh-Brg-10.53±0.08,t=5.292,P<0.01),HIF-1α(con 1.00±0 vs.sh-Brg-10.61±0.07,t=5.380,P<0.01)and VEGF-A(con 1.00±0 vs.sh-Brg-10.38±0.10,t=6.169,P<0.01)was decreased compared with the Lenti-con group in normoxia on mRNA level.Meanwhile,knockdown Brg-1 also reduced the expression of Brg-1(con 1.00±0 vs.sh-Brg-10.07±0.02,t=44.680,P<0.01),HIF-1α(con 1.00±0 vs.sh-Brg-10.21±0.05,t=14.280,P<0.01),VEGF-A(con 1.00±0 vs.sh-Brg-10.15±0.06,t=13.960,P<0.01)in hypoxia on mRNA level.The deficiency of brg-1 reduced angiogenesis of colorectal cancer in vitro(Normoxia,con 55.67±7.22 vs.sh-Brg-122.33±5.36,t=3.706,P<0.05;Hypoxia,con 66.33±7.62 vs.sh-Brg-135.68±4.91,t=3.382,P<0.05).Conclusion The low expression of Brg-1 in LoVo cell line can inhibit angiogenesis via HIF-1α.