CpG hypermethylation of human four-and-a-half LIM domains 1 contributes to migration and invasion activity of human bladder cancer.
We previously reported a simple technique that combines microarray data from clinical bladder cancer (BC) specimens with those from a BC cell line (BOY) treated with a pharmacologic demethylating agent (5-aza-dC). We focused on the human four-and-a-half LIM domains 1 (FHL1) gene which was selected on the basis of previous microarray data analysis. Because LIM domains provide protein-protein binding interfaces, FHL genes play an important role in cellular events, such as focal adhesion and differentiation, by interacting with the target protein as either a repressor or activator. We hypothesized that inactivation of the FHL1 gene through CpG methylation contributes to cell viability including migration and invasion activity of human BC. After 5-aza-dC treatment, the expression levels of FHL1 mRNA transcript markedly increased in all cell lines tested, as shown by real-time reverse transcription-polymerase chain reaction (RT-PCR). The methylation index of FHL1 in our samples was significantly higher in 70 BC specimens than in 10 normal bladder epithelium (NBE) specimens (63.9+/-25.5 and 0.3+/-0.2, respectively; p=0.0066). Conversely, FHL1 mRNA expression was significantly lower in the BC specimens than in the NBE ones (0.331+/-0.12 and 2.498+/-0.61, respectively; p=0.0011). In addition, significant inhibitions of wound healing (45.78+/-6.2, and 100+/-0, respectively; p=0.009) and of cell invasion (18.5+/-2.3 and 95.2+/-2.4, respectively; p=0.02) were observed in stable FHL1-transfected cells than in the control BC cells. In conclusion, we found that the mechanism of FHL1 down-regulation in BC is through CpG hypermethylation of the promoter region. FHL1 gene inactivation by CpG hypermethylation may thus contribute to migration and invasion activity of BC.
Matsumoto
Mitsugi
M
Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
Kawakami
Kazumori
K
Enokida
Hideki
H
Toki
Kazuki
K
Matsuda
Ryoichiro
R
Chiyomaru
Takeshi
T
Nishiyama
Kenryu
K
Kawahara
Kazuya
K
Seki
Naohiko
N
Nakagawa
Masayuki
M
D000368:Aged; D000369:Aged, 80 and over; D001374:Azacitidine; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D018899:CpG Islands; D019175:DNA Methylation; D004791:Enzyme Inhibitors; D005260:Female; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D060588:LIM Domain Proteins; D008297:Male; D008875:Middle Aged; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000378:metabolism*
D004791:Enzyme Inhibitors; C101149:FHL1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D009124:Muscle Proteins; D001374:Azacitidine