Fig 2: Primary data for surgical samples from a 68-year-old woman (study patient). a. Down-regulation of the COL1A1 gene was seen in the tumor tissue compared with the adjacent noncancerous liver tissue (146 bp). Reverse transcriptase polymerase chain reaction (RT-PCR) for ß-actin was performed to normalize the quantity of cDNA. b Copy number analysis of chromosome 17. There was no deletion or amplification at the COL1A1 gene locus (17q21.33). c Immunohistochemical staining of COL1A1 protein showed that tumor tissue components showed almost no staining compared with adjacent noncancerous tissue components (200×).

Fig 3: Analysis of COL1A1 methylation and expression. a Promoter methylation status of the study patient’s samples was examined. Methylation-specific PCR (MSP) and unmethylation-specific PCR (UMSP) were performed. Only tumor tissue had promoter methylation. b Promoter methylation status of the COL1A1 gene six hepatcellular carcinoma (HCC) cell lines. Complete methylation was detected in the cell line HLE; partial methylation in HLF, HuH2, and SK-Hep1; and complete unmethylation in Hep3B and HuH7. cCOL1A1 expression was reactivated in HLE, HLF, HuH2, and SK-Hep1 by 5-aza-2'-deoxycytidine (5-aza-dC) treatment. ß-Actin was used as the normalization gene. d COL1A1 protein expression was confirmed by western blotting. Very weak or no band was detected in the cell lines with positive promoter methylation. ß-Actin was used as the normalization gene.

Fig 4: Overall survival curves for down-regulated and up-regulated cases of COL1A1 mRNA expression in tumor tissues. Red line: down-regulated (n = 13). Blue line: up-regulated (n = 35). According to the log-rank test, down-regulated cases were significantly correlated with poor overall survival (P = 0.013).