Natural antisense transcript-targeted regulation of inducible nitric oxide synthase mRNA levels.
Natural antisense transcripts (asRNAs) are frequently transcribed from mammalian genes. Recently, we found that non-coding asRNAs are transcribed from the 3' untranslated region (3'UTR) of the rat and mouse genes encoding inducible nitric oxide synthase (iNOS), which catalyzes the production of the inflammatory mediator nitric oxide. The iNOS asRNA stabilizes iNOS mRNA by interacting with the mRNA 3'UTR. Furthermore, single-stranded 'sense' oligonucleotides corresponding to the iNOS mRNA sequence were found to reduce iNOS mRNA levels by interfering with mRNA-asRNA interactions in rat hepatocytes. This method was named natural antisense transcript-targeted regulation (NATRE) technology. In this study, we detected human iNOS asRNA expressed in hepatocarcinoma and colon carcinoma tissues. The human iNOS asRNA harbored a sequence complementary to an evolutionarily conserved region of the iNOS mRNA 3'UTR. When introduced into hepatocytes, iNOS sense oligonucleotides that were modified by substitution with partial phosphorothioate bonds and locked nucleic acids or 2'-O-methyl nucleic acids greatly reduced levels of iNOS mRNA and iNOS protein. Moreover, sense oligonucleotides and short interfering RNAs decreased iNOS mRNA to comparable levels. These results suggest that NATRE technology using iNOS sense oligonucleotides could potentially be used to treat human inflammatory diseases and cancers by reducing iNOS mRNA levels.
Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, Japan.
Emi
E
Yoshigai
Takafumi
T
Hara
Yoshiro
Y
Araki
Yoshito
Y
Tanaka
Masaharu
M
Oishi
Katsuji
K
Tokuhara
Masaki
M
Kaibori
Tadayoshi
T
Okumura
A-Hon
AH
Kwon
Mikio
M
Nishizawa
D020413:3' Untranslated Regions; D000818:Animals; D003110:Colonic Neoplasms / Q000737:chemistry / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000737:chemistry / Q000378:metabolism; D008297:Male; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D016372:RNA, Antisense / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D018709:Statistics, Nonparametric
D020413:3' Untranslated Regions; D016372:RNA, Antisense; D012333:RNA, Messenger; D052247:Nitric Oxide Synthase Type II