Effects of antisense oligonucleotide targeting osteopontin on invasion and metastasis of colon carcinoma cell line HT-29 in vitro under moderate hypoxia

Abstract

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 microporous 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 ceils 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 cells were exposed to moderate hypoxia. Key words: Colon carcinoma; Antisense oligonueleotide; Osteopontin; Malignant phenotype