Hepatocellular carcinoma (HCC) is one of the most common cancers world-wide but the molecular mechanisms that underlie hepatocarcinogenesis are not fully determined. On the same surgical sample with HCC, we performed microarray-based gene expression profiling and karyotype analysis using a single nucleotide polymorphism (SNP) array. In addition, quantitative real-time reverse transcription polymerase chain reaction (PCR), methylation specific PCR (MSP) and immunohistochemical staining were conducted using specimens from 48 patients with HCC. Gene expression profiling showed the expression of fibulin 1 (FBLN1), located on 22q13, to be decreased in tumor tissue. Karyotype analysis revealed no loss of heterozygosity (LOH) since deletions were not detected in 22q, and one of the SNPs on 22q13 showed AB genotype in both cancerous tissue and in corresponding noncancerous tissue, indicating retention of heterozygosity. Quantitative real-time PCR showed FBLN1 mRNA levels in cancerous tissues to be significantly decreased compared with that in corresponding noncancerous tissues. The immunohistochemical staining results were consistent with both gene expression profiling and quantitative PCR data. Twenty-four out of 48 HCCs gave a positive result in MSP. Moreover, promoter hypermethylation of FBLN1 was significantly associated with advanced stage HCC, multiple tumors and increased tumor size. Our results indicated that FBLN1 is a novel candidate of tumor suppressor gene and that promoter hypermethylation of FBLN1 is associated with tumor progression in HCC. © 2011 Wiley-Liss, Inc.
Hepatocellular carcinoma (HCC) is one of the top five causes of cancer-related deaths worldwide. Recent developments in the treatment of HCC remain insufficient to cure unresectable disease or to prevent HCC. Consistent efforts are, therefore, needed to deepen understanding of pathogenesis of the disease. Genome-wide gene expression profile analyses can now detect various candidate genes that are modified by HCC. We have developed a new technique to identify tumor suppressor genes, triple-combination array analysis, which combines gene expression profiles, single nucleotide polymorphism and methylation arrays to identify genes with altered expression. Using HCC tissue samples, triple-combination array analysis was performed to identify a candidate tumor suppressor gene. Subsequently, samples from 48 HCC patients were subjected to quantitative polymerase chain reaction (qPCR) and methylation-specific PCR to further elucidate clinical relevance of the gene. Estrogen receptor 1 (ESR1) was detected as a candidate tumor suppressor gene. Of the 48 clinical samples, 40 (83.3%) showed ESR1 promoter hypermethylation. In 24 (50%) HCC samples, the expression levels of the ESR1 gene was decreased by >90%. The decreased expression was significantly related to high liver damage score, pathological invasion of the intrahepatic portal vein, the size of tumor (>3 cm in diameter) and hepatitis B virus infection. The present study represents another example that triple-combination array is a convenient technique for detecting genes with altered expression in disease. The ESR1 gene was identified as a candidate tumor suppressor gene in HCC and further validation is warranted.
BackgroundHepatocellular 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.MethodsTriple-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.ResultsExpression 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).ConclusionsTriple-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.
BackgroundTo identify genes associated with hepatocellular carcinoma (HCC) pathogenesis, we developed a triple combination array strategy comprising methylation, gene expression, and single nucleotide polymorphism (SNP) array analysis.MethodsSurgical specimens obtained from a 68-year-old female HCC patient were analyzed by triple combination array, and identified Dynamin 3 (DNM3) as a candidate tumor suppressor gene in HCC. Subsequently, samples from 48 HCC patients were evaluated for DNM3 methylation and expression status using methylation specific polymerase chain reaction (PCR; MSP) and semi-quantitative reverse transcriptase (RT)-PCR, respectively. The relationship between clinicopathological factors and DNM3 methylation status was also investigated.ResultsDNM3 was shown to be hypermethylated (methylation value 0.879, range 0–1.0) in cancer tissue compared with adjacent normal tissue (0.213) by methylation array in the 68-year-old female patient. Expression arrays revealed decreased expression of DNM3 in cancerous tissue. SNP arrays revealed that the copy number of chromosome 1q24.3, in which DNM3 resides, was normal. MSP revealed hypermethylation of the DNM3 promoter region in 33 of 48 tumor samples. A trend toward decreased DNM3 expression was observed in patients with DNM3 promoter methylation (P = 0.189). Furthermore, patients with reduced expression of DNM3 in tumor tissues exhibited worse prognosis with decreased disease specific survival compared to patients without decreased expression (P = 0.014).ConclusionThe present study indicates that a triple combination array strategy is an effective method to detect novel genes related to HCC. We propose that DNM3 is a tumor suppressor gene in HCC.
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide and its prognosis is poor. Novel targets for treating recurrence and progression along with associated biomarkers are urgently required. In this study, the expression and regulatory mechanism of DENN/MADD domain containing 2D (DENND2D) were investigated in an attempt to identify a tumor suppressor gene for HCC regulated by silencing through promoter hypermethylation. The levels of DENND2D expression in HCC cell lines and surgical specimens were determined using a quantitative polymerase chain reaction assay and the relationship between the expression levels of DENND2D mRNA and clinicopathological factors was evaluated. The expression and distribution of DENND2D were determined using immunohistochemistry. DNA methylation analysis was performed to determine the regulatory mechanisms of DENND2D expression in HCC. Most HCC cell lines (89%) and surgical specimens (78%) expressed lower levels of DENND2D mRNA compared with normal liver tissue. In contrast, there was no significant difference in the expression levels of DENND2D mRNA between normal tissues of HCC patients with and without cirrhosis. The expression patterns of DENND2D protein and mRNA were consistent. Patients with significantly lower levels of DENND2D mRNA in HCC tissues had remarkably earlier recurrences after hepatectomy and their prognosis worsened. The DENND2D promoter was methylated in eight out of nine HCC cell lines and DNA demethylation reactivated DENND2D mRNA expression. Hypermethylation of DENND2D was frequently detected in HCC tissues (75%) and was significantly associated with downregulation of DENND2D mRNA expression. DENND2D is a candidate tumor suppressor gene that is inactivated by promoter hypermethylation in patients with HCC and may serve as a novel biomarker of early recurrence of HCC.
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