Identification of the collagen type 1 α 1 gene (COL1A1) as a candidate survival-related factor associated with hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related death especially among Asian and African populations. It is urgent that we identify carcinogenesis-related genes to establish an innovative treatment strategy for this disease.

Methods: Triple-combination array analysis was performed using one pair each of HCC and noncancerous liver samples from a 68-year-old woman. This analysis consists of expression array, single nucleotide polymorphism array and methylation array. The gene encoding collagen type 1 alpha 1 (COL1A1) was identified and verified using HCC cell lines and 48 tissues from patients with primary HCC.

Results: Expression array revealed that COL1A1 gene expression was markedly decreased in tumor tissues (log2 ratio -1.1). The single nucleotide polymorphism array showed no chromosomal deletion in the locus of COL1A1. Importantly, the methylation value in the tumor tissue was higher (0.557) than that of the adjacent liver tissue (0.008). We verified that expression of this gene was suppressed by promoter methylation. Reactivation of COL1A1 expression by 5-aza-2'-deoxycytidine treatment was seen in HCC cell lines, and sequence analysis identified methylated CpG sites in the COL1A1 promoter region. Among 48 pairs of surgical specimens, 13 (27.1%) showed decreased COL1A1 mRNA expression in tumor sites. Among these 13 cases, 10 had promoter methylation at the tumor site. The log-rank test indicated that mRNA down-regulated tumors were significantly correlated with a poor overall survival rate (P = 0.013).

Conclusions: Triple-combination array analysis successfully identified COL1A1 as a candidate survival-related gene in HCCs. Epigenetic down-regulation of COL1A1 mRNA expression might have a role as a prognostic biomarker of HCC.

Figure 1

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 β-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×).

Figure 2

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. β-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. β-Actin was used as the normalization gene.

Figure 3

Direct sequencing of bisulfite-treated HCC cell lines. The location was between −12 and +35 bp from the transcription initiation site. All CG dinucleotides were almost unmethylated (blue circles) in Hep3B. In contrast, all CG dinucleotides were completely methylated (red circles) in HLE.

Figure 4

Promoter methylation status in 48 tumor tissues and matched noncancerous liver tissues. a A total of 20 tumor tissues and 11 noncancerous tissues showed promoter methylation. b MSP and UMSP results of two representative cases. c The 48 cases were divided into13 COL1A1 expression down-regulated cases and 35 up-regulated cases in tumor tissues. Promoter methylated cases were indicated by red lines. Promoter methylation and the mRNA expression pattern were significantly correlated (P = 0.002).

Figure 5

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).