摘要
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细胞侵袭转移潜能等的影响，探讨OPN与微缺氧诱导的肿瘤恶性表型的关系。方法合成ASODN，以Lipofectamine为载体，将其转染入微缺氧下高表达OPN的HT-29细胞。RT—PCR和Western blot分别检测转染细胞微缺氧下OPN mRNA和蛋白的表达；Transwell侵袭试验判定其侵袭游走能力；明胶酶谱分析检测分泌的MMP-2／9活性变化。结果微缺氧诱导的OPN mRNA和蛋白表达被靶向OPN的ASODN特异性地抑制，表达量分别是对照组的31．5％和35．4％。ASODN组HT-29细胞穿过微孔膜的细胞数仅为（52．6±3．8）个；明胶酶活性下降71％，与各对照组比较，差异有统计学意义（P〈0．01）。结论靶向OPN的ASODN明显抑制微缺氧诱导的OPN mRNA和蛋白表达，并显著下调微缺氧诱导的细胞分泌MMP2／9的活性及其侵袭游走能力。OPN在微缺氧促进肿瘤向恶性表型转化中起着重要作用。
Objective To study the effects of antisense oligonucleotide （ASODN） targeting osteopontin on the invasion of HT-29 cells under moderate hypoxia, and investigate correlation between osteopontin and malignant phenotype induced by moderate hypoxia. Methods ASODN targeting osteopontin was synthesized with a phosphorothioate backbone. By Lipofectamine, ASODN was transfected into HT-29 cells with high expression of osteopontin induced by moderate hypoxia. The osteopontin mRNA and protein levels in HT-29 cells transfected with ASODN were detected by RT-PCR and Western blot respectively. Reaction to a hypoxic environment was determined via invasion across a Matrigel-coated Transwell filter. The activity of MMP-2 and MMP-9 was assessed using gelatin zymography. Results ASODN targeting osteopontin could selectively and significantly down-regulate the levels of osteopontin mRNA and protein, with 31.5% and 35.4% of the controls respectively. The number of HT-29 cells migrating to the micro- porous membrane in ASODN group was only 52.6 _＋ 3.8. ASODN targeting osteopontin could suppress significantly the MMP2/9 activities down to 71% in HT-29 cells exposed to moderate hypoxia. Conclusion ASODN targeting osteopontin could down-regulate the osteopontin mRNA and protein levels in HT-29 cells exposed to moderate hypoxia. The invasive capacity and MMP2/9 activities previously induced by moderate hypoxia could be inhibited when osteopontin was suppressed by ASODN. It suggested that osteopontin might play a key role in malignant phenotype when HT-29 ceils were exposed to moderate hypoxia.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2009年第9期1163-1165,共3页
Chinese Journal of Experimental Surgery
关键词
结肠癌
反义寡核苷酸
骨桥蛋白
恶性表型
Colon carcinoma
Antisense oligonucleotide
Osteopontin
Malignant phenotype
