A noncoding RNA gene on chromosome 10p15.3 may function upstream of hTERT.
BACKGROUND
We attempted to clone candidate genes on 10p 14-15 which may regulate hTERT expression, through exon trapping using 3 BAC clones covering the region. After obtaining 20 exons, we examined the function of RGM249 (RGM: RNA gene for miRNAs) we cloned from primary cultured human hepatocytes and hepatoma cell lines. We confirmed approximately 20 bp products digested by Dicer, and investigated the function of this cloned gene and its involvement in hTERT expression by transfecting the hepatoma cell lines with full-length dsRNA, gene-specific designed siRNA, and shRNA-generating plasmid.


RESULTS
RGM249 showed cancer-dominant intense expression similar to hTERT in cancer cell lines, whereas very weak expression was evident in human primary hepatocytes without telomerase activity. This gene was predicted to be a noncoding precursor RNA gene. Interestingly, RGM249 dsRNA, siRNA, and shRNA inhibited more than 80% of hTERT mRNA expression. In contrast, primary cultured cells overexpressing the gene showed no significant change in hTERT mRNA expression; the overexpression of the gene strongly suppressed hTERT mRNA in poorly differentiated cells.


CONCLUSIONS
These findings indicate that RGM249 might be a microRNA precursor gene involved in the differentiation and function upstream of hTERT.
Miura
Norimasa
N
Division of Pharmacotherapeutics, Department of Pathophysiological and Therapeutic Science, Faculty of Medicine, Tottori University, Yonago, Tottori 683-8503, Japan. mnmiura@hotmail.com
Sato
Reina
R
Tsukamoto
Tomoe
T
Shimizu
Mika
M
Kabashima
Hiroko
H
Takeda
Miho
M
Takahashi
Shunsaku
S
Harada
Tomomi
T
West
James E
JE
Drabkin
Harry
H
Mejia
Jose E
JE
Shiota
Goshi
G
Murawaki
Yoshikazu
Y
Virmani
Arvind
A
Gazdar
Adi F
AF
Oshimura
Mitsuo
M
Hasegawa
Junichi
J
D001483:Base Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D002879:Chromosomes, Human, Pair 10; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism
D035683:MicroRNAs; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019098:Telomerase