骨桥蛋白反义寡核苷酸对体外微缺氧诱导的肿瘤新生血管的影响

Effects of antisense oligonucleotide targeting osteopontin on tumor angiogenesis in vitro under moderate hypoxia

目的观察靶向骨桥蛋白(O PN)的反义寡核苷酸(ASO D N)对微缺氧下H T-29细胞体外促进新生血管形成能力的影响,探讨O PN与微缺氧诱导的肿瘤新生血管的关系。方法合成靶向O PN的ASO D N,以Lipofectam ine为载体,将其转染入微缺氧下高表达O PN的H T-29细胞。R T-PC R和W estern blot分别检测转染细胞微缺氧下O PN m R N A和蛋白的表达;明胶酶谱分析检测分泌的M M P-2/M M P-9活性变化;内皮细胞小管形成试验检测体外促进新生血管形成的能力。提取胞核蛋白,W estern blot检测胞核内N F-κB/p65蛋白表达。结果微缺氧诱导的O PN m R N A和蛋白表达被靶向O PN的ASO D N特异性地抑制,表达量分别是对照组的35.4%和31.5%。ASO D N组H T-29细胞明胶酶活性下降71.0%,内皮细胞形成管状结构的数仅为(12.4±1.8);胞核内的N F-κB/p65蛋白表达下调了61.2%。与各对照组相比,差异具有统计学意义(P<0.01)。结论靶向O PN的ASO D N明显抑制微缺氧诱导的O PN m R N A和蛋白表达,显著下调微缺氧诱导的H T-29细胞分泌M M P-2/M M P-9的活性,显著拮抗新生血管形成。阻抑O PN的表达,胞核内N F-κB/p65表达、M M P-2/M M P-9的活性随之下调,提示N F-κB和M M P-2/M M P-9也可能参与微缺氧诱导的恶性转化,并且是O PN的下游调控基因。

【Objective】 To study the effects of antisense oligonucleotide（ASODN） targeting osteopontin on the ability of tumor angiogenesis of HT-29 cells in vitro under moderate hypoxia.To investigate correlation between osteopontin and tumor angiogenesis induced by moderate hypoxia.【Methods】 ASODN targeting osteopontin was synthesized with a phosphorothioate backbone.Mediating by Lipofectamine,ASODN were transfected into HT-29 cells with high expression of osteopontin induced by moderate hypoxia.The osteopontin mRNA and protein levels of HT-29 cells treated with ASODN were detected by RT-PCR and western blot,respectively.Similarly,the expression of NF-κB/p65 in nucleus was detected by western blot after extraction of nucleic protein.The activity of MMP-2 and MMP-9 was assessed using gelatin zymography.Tube formation of human umbilical vein endothelial cells（HUVECs） were adopted to evaluate the ability of tumor angiogenesis in vitro.【Result】 ASODN targeting osteopontin could selectively and significantly down-regulate the levels of osteopontin mRNA,protein and NF-κB/p65 protein in nucleus of HT-29 cells exposed to moderate hypoxia,with 35.4%,31.5% and 38.8% of the control respectively.ASODN targeting osteopontin could suppress significantly the MMP2/MMP-9 activities down to 71% in HT-29 cells exposed to moderate hypoxia.The numbers of tubes formed by HUVECs in ASODN group were only（12.4±2.8）.The above results showed a significant difference between ASODN group and other controls.【Conclusion】 ASODN targeting osteopontin could down-regulate the osteopontin mRNA and protein levels in HT-29 cells exposed to moderate hypoxia.The angiogenesis and MMP-2/MMP-9 activities and the levels of NF-κB/p65 in nucleus previously induced by moderate hypoxia could be inhibited when osteopontin was suppressed by ASODN.It also suggested that NF-κB and MMP-2/MMP-9 could involve malignant phenotype induced by moderate hypoxia and could be regulatory downstream genes of osteopontin.