CpG hypermethylation of collagen type I alpha 2 contributes to proliferation and migration activity of human bladder cancer.
In our microarray screening of methylated genes in bladder cancer (BC), the collagen type 1 alpha 2 (COL1A2) gene was the most up-regulated among the 30,144 genes screened. We hypothesize that inactivation of the COL1A2 gene through CpG methylation contributes to proliferation and migration activity of human BC. We subjected a bladder cancer cell line (BOY) and 67 BC specimens and 10 normal bladder epitheliums (NBEs) to conventional or real-time methylation quantitative polymerase chain reaction (PCR) and to real-time reverse transcriptase (RT)-PCR. We also established a stable COL1A2 transfectant for evaluating cell proliferation and migration activity. After 5-aza-dC treatment, the expression levels of COL1A2 mRNA transcript markedly increased in BOY. Our cell proliferation assays consistently demonstrated growth inhibition in the COL1A2 transfectant compared with control and wild-type BOY cells (p<0.0001). Wound healing assays also showed significant wound healing inhibition in the COL1A2 transfectant compared to the counterparts (p=0.0016). We demonstrated by bisulfite DNA sequencing that the promoter hypermethylation of COL1A2 was a frequent event in clinical BCs. The methylation index of COL1A2 was significantly higher in the 67 BCs than in the 10 NBEs (p=0.0011). Conversely, COL1A2 mRNA transcript was significantly lower in the BCs than in the NBEs (p=0.0052). The mechanism of COL1A2 down-regulation in BC is through CpG hypermethylation of the promoter region. COL1A2 gene inactivation through CpG hypermethylation may contribute to proliferation and migration activity of BC.
Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8520, Japan.
Katsuhisa
K
Mori
Hideki
H
Enokida
Ichiro
I
Kagara
Kazumori
K
Kawakami
Takeshi
T
Chiyomaru
Shuichi
S
Tatarano
Kazuya
K
Kawahara
Kenryu
K
Nishiyama
Naohiko
N
Seki
Masayuki
M
Nakagawa
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation; D003094:Collagen / Q000235:genetics* / Q000378:metabolism; D024042:Collagen Type I; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D000077209:Decitabine; D015536:Down-Regulation; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D008297:Male; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000235:genetics / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D001743:Urinary Bladder / Q000378:metabolism / Q000473:pathology; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000473:pathology*
D014408:Biomarkers, Tumor; D024042:Collagen Type I; D012333:RNA, Messenger; D000077209:Decitabine; D003094:Collagen; D001374:Azacitidine