HIPK2调控COX-2及β-catenin表达对人结直肠癌细胞VEGF表达的影响  

HIPK2 reduces VEGF expression by regulating expression of COX-2 and β-catenin in human colorectal cancer cells

作　　者：胡强 周利红[2] 刘宣[2] 隋华[2] 付晓伶[2] 颜琳琳[2] 任建琳[3] 李琦[2] 

机构地区：[1]上海市徐汇区大华医院普外科,上海市200237 [2]上海中医药大学附属曙光医院肿瘤科,上海市201203 [3]上海市中医医院肿瘤科,上海市200071

出　　处：《世界华人消化杂志》2014年第9期1266-1274,共9页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;Nos.81303103;81303106;上海市科委基金资助项目;Nos.13140902500;13ZR1462200;上海市教委创新基金资助项目;Nos.12ZZ118;12YZ058;上海市卫生局基金资助项目;Nos.2010161;2011ZJ030;20124048;上海市徐汇区医学科研基金资助项目;No.SHXH201004~~

摘　　要：目的:研究同源结构域相互作用蛋白激酶2(homeodomain-interacting protein kinases 2,HIPK2)基因重组质粒对HCT-116细胞环氧合酶2(cyclooxygenase 2,COX-2)及β-catenin表达的影响,探讨HIPK2基因对人结直肠癌细胞血管新生因子表达的调节作用.方法:采用脂质体将pEGFP-N3和pEGFP-N3-HIPK2质粒转染人结直肠癌细胞,分为正常组、载体质粒组和HIPK2质粒组,ELISA法检测细胞培养液中血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达,Western blot检测β-catenin蛋白表达情况.在上述3组细胞中同时转染含COX-2基因启动子质粒pGL3-basic-COX-2后,双荧光素酶活性检测COX-2启动子的表达,Real-time PCR检测COX-2 mRNA的表达水平.结果:HCT-116细胞中pEGFP-N3-HIPK2质粒组的荧光表达量与载体质粒pEGFP-N3组的表达量基本一致,两组相比无明显差异.转染pEGFP-N3-HIPK2质粒后,HCT-116细胞内VEGF的表达量由对照组的857.54p g/m L±65.04 p g/m L降低至368.32 p g/m L±98.82 pg/mL,两组相比具有统计学意义(P<0.05);COX-2启动子活性由空载体组的266.407×10-5±40.902×10-5下降至75.467×10-5±18.666×10-5,两组相比具有统计学意义(P<0.05);COX-2 mRNA的相对表达量由空载体组的3.48×10-4±0.64×10-4下调至1.07×10-4±0.32×10-4,两组相比具有统计学意义(P<0.05).同时,Western blot结果显示,HIPK2可明显降低细胞β-catenin的蛋白表达,上调细胞内p-β-catenin蛋白的表达量.结论:pEGFP-N3-HIPK2可明显上调HCT-116细胞HIPK2的表达,显著下调人结直肠癌细胞COX-2的转录表达及VEGF的表达水平,其作用机制可能与其抑制细胞COX-2及β-catenin表达,调节β-catenin-COX-2信号转导有关.AIM： To study the effects of overexpressionof homeodomain-interacting protein kinase 2 （HIPK2） on the expression of cyclooxygenase 2 （COX-2） and （3-catenin in HCT-116 cells, and to explore the regulatory effect of the HIPK2 gene on angiogenic factor expression in human colorectal cancer cells. METHODS： The pEGFP-N3 vector or pEGFP- N3-HIPK2 plasmid was transfected into human colorectal cancer HCT-116 cells using Lipo- fectamine 2000, and untransfected cells were used as normal controls. The expression of VEGF （vas- cular endothelial growth factor） in cell culture me- dium was detected by ELISA assay, and the protein expression of β-catenin was tested by Western blot. The COX-2 promoter containing plasmid pGL3- basic-COX-2 was co-transfected with pRL-SV40 in the three groups above. COX-2 promoter activity was detected by dual luciferase activity assay, and COX-2 mRNA level was determined by real-time PCR. RESULTS： The pEGFP-N3-HIPK2 group and pEGFP-N3 group had the same fluorescent level, and there was no statistically significant difference in HCT-116 cells. The VEGF expres- sion was significantly inhibited （857.54 pg/mL ± 65.04 pg/mL vs 368.32 pg/mL ± 98.82 pg/mL, P 〈 0.05） after pEGFP-N3-HIPK2 plasmid transfec- tion. COX-2 promoter activity and mRNA ex- pression were significantly lower in the pEGFP- N3-HIPK2 group than in the pEGFP-N3 group （75.467×10-5 ± 18.666×10-5 vs 266.407×10-5 ± 40.902×10-5, 1.07×10-4 ± 0.32×10-4 vs 3.48×10-4 ± 0.64×10-4 P 〈 0.05 for both）. HIPK2 could inhibit the pro- tein expression of β-catenin but up-regulate p-β- catenin expression. CONCLUSION： pEGFP-N3-HIPK2 transfection significantly increased the expression of HIPK2, and reduced the transcription of the COX2 gene and VEGF level in human colorectal cancer HCT-116 cells. The mechanisms may be related to the inhibition of COX-2 and β-catenin expres- sion in cells, and regulation ofβ-catenin-COX-2 signal transduction.

关 键 词：同源结构域相互作用蛋白激酶2 血管内皮生长因子 结直肠癌 血管新生 环氧合酶2 

分 类 号：R735.3[医药卫生—肿瘤]