反义寡核苷酸对微缺氧下人结肠癌细胞HT-29骨桥蛋白表达的影响

摘要

Objective To study the effects of antisense oligonucleotide (ASODN) targeting osteopontin on its expression of HT-29 cells under moderate hypoxia and to lay the foundation for investigating correlation between osteopontin and malignant phenotype induced by moderate hypoxia.Methods ASODN targeting osteopontin were synthesized with a phosphorothioate backbone.Mediated by Lipofectamine, ASODN were transfected into HT-29 cells with high expression of osteopontin induced by moderate hypoxia.The specificity and dose-effect relationship between ASODN and osteopontin protein were examined by Western blot.Osteopontin mRNA levels of HT-29 cells treated with ASODN were detected by RT-PCR.Result ASODN targeting osteopontin inhibited the expression of osteopontin protein in a dose-dependent manner.However, this tendency between levels of 10? mol/L and of 20? mol/L declined and showed no significant difference.ASODN targeting osteopontin could selectively and significantly down-regulate the levels of osteopontin protein and mRNA, with 35.4％ and 31.5％ of the controls respectively.Conclusion ASODN targeting osteopontin could down-regulate the osteopontin mRNA and protein levels in HT-29 cells exposed to moderate hypoxia.%目的 观察靶向骨桥蛋白(OPN)的反义寡核苷酸(ASODN)对微缺氧下HT-29细胞骨桥蛋白表达的影响,为探讨OPN与微缺氧诱导的肿瘤恶性表型的关系奠定基础.方法 合成ASODN,以Lipofectamine为栽体,将其转染入微缺氧下高表达OPN的HT-29细胞.Western blot行OPN ASODN抑制OPN蛋白表达的量效关系和特异性分析.RT-PCR检测转染细胞微缺氧下OPNmRNA的表达.结果 ASODN组OPN蛋白表达较各对照组明显下调(P<0.01),其下调的幅度随OPN ASODN浓度升高而增强,但这种增强趋势在浓度为10umol/L和20umol/L之间已不明显(P>0.05).微缺氧诱导的OPN蛋白和mRNA表达被靶向OPN的ASODN特异性地抑制,表达量分别是对照组的35.4%争31.5%.结论 靶向OPN的ASODN明显抑制微缺氧诱导的OPNmRNA和蛋白表达.