Aberrant DNA methylation profile and frequent methylation of KLK10 and OXGR1 genes in hepatocellular carcinoma.
Investigating aberrant DNA methylation in the cancer genome may identify genes that play an important role in tumor progression. In this study, we combined differential methylation hybridization and a CpG microarray platform to characterize methylation profiles and identify novel candidate genes associated with hepatocellular carcinoma (HCC). The genomic DNA of 21 paired adjacent normal and HCC samples was used, and results were analyzed by hierarchical clustering. Twenty-seven hypermethylated candidates and 38 hypomethylated candidates were obtained. Six candidate genes from the hypermethylated group were validated by combined bisulfite restriction analysis; two genes, human kallikrein 10 gene (KLK10) and oxoglutarate (alpha-ketoglutarate) receptor 1 gene (OXGR1), were further analyzed by bisulfite sequencing. The DNA hypermethylation status of KLK10 and OXGR1 were subsequently examined in HCC cell lines and clinical samples using methylation-specific PCR. In 49 HCC samples, 46 (94%) showed that at least one of these two genes was highly methylated. Moreover, KLK10 and OXGR1 mRNA levels were inversely correlated (r = -0.435 and -0.497, P < 0.05) with DNA methylation as examined in paired adjacent normal and tumor samples. Statistical analyses further indicated that KLK10 hypermethylation was significantly associated with cirrhosis (P = 0.042) and HCV infection (P = 0.017) as well as inversely associated with HBV infection (P = 0.023). Furthermore, restoration of KLK10 and OXGR1 expression reduced the ability of anchorage-independent growth, and sensitized HCC cells to doxorubicin- or 5-fluorouracil-induced cytotoxicity. Our results suggest that the hypermethylated KLK10 and OXGR1 are frequent in HCC and may be useful as markers for clinical application.
Lu
Chang-Yi
CY
Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan.
Hsieh
Sen-Yung
SY
Lu
Yen-Jung
YJ
Wu
Chi-Sheng
CS
Chen
Lih-Chyang
LC
Lo
Shao-Jung
SJ
Wu
Cheng-Tao
CT
Chou
Min-Yuan
MY
Huang
Tim Hui-Ming
TH
Chang
Yu-Sun
YS
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D003114:Colony-Forming Units Assay; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007610:Kallikreins / Q000235:genetics*; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics*; D018048:Receptors, Purinergic P2; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
C502602:OXGR1 protein, human; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled; D018048:Receptors, Purinergic P2; C100936:KLK10 protein, human; D007610:Kallikreins