Detection of doublecortin domain-containing 2 (DCDC2), a new candidate tumor suppressor gene of hepatocellular carcinoma, by triple combination array analysis

Yoshikuni Inokawa,Hiroyuki Sugimoto,Shin Takeda,Masamichi Hayashi,Yoko Nishikawa,Yasuhiro Kodera,Suguru Yamada,Tsutomu Fujii,Mitsuhiro Hishida,Shuji Nomoto,Mitsuro Kanda

doi:10.1186/1756-9966-32-65

Yoshikuni Inokawa, Hiroyuki Sugimoto + Show 9 more

Open Access

https://doi.org/10.1186/1756-9966-32-65

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Abstract

BackgroundTo detect genes correlated with hepatocellular carcinoma (HCC), we developed a triple combination array consisting of methylation array, gene expression array and single nucleotide polymorphism (SNP) array analysis.MethodsA surgical specimen obtained from a 68-year-old female HCC patient was analyzed by triple combination array, which identified doublecortin domain-containing 2 (DCDC2) as a candidate tumor suppressor gene of HCC. Subsequently, samples from 48 HCC patients were evaluated for their DCDC2 methylation and expression status using methylation specific PCR (MSP) and semi-quantitative reverse transcriptase (RT) PCR, respectively. Then, we investigated the relationship between clinicopathological factors and methylation status of DCDC2.ResultsDCDC2 was revealed to be hypermethylated (methylation value 0.846, range 0–1.0) in cancer tissue, compared with adjacent normal tissue (0.212) by methylation array in the 68-year-old female patient. Expression array showed decreased expression of DCDC2 in cancerous tissue. SNP array showed that the copy number of chromosome 6p22.1, in which DCDC2 resides, was normal. MSP revealed hypermethylation of the promoter region of DCDC2 in 41 of the tumor samples. DCDC2 expression was significantly decreased in the cases with methylation (P = 0.048). Furthermore, the methylated cases revealed worse prognosis for overall survival than unmethylated cases (P = 0.048).ConclusionsThe present study indicates that triple combination array is an effective method to detect novel genes related to HCC. We propose that DCDC2 is a tumor suppressor gene of HCC.

Highlights

To detect genes correlated with hepatocellular carcinoma (HCC), we developed a triple combination array consisting of methylation array, gene expression array and single nucleotide polymorphism (SNP) array analysis
SNP, and methylation-arrays To identify novel tumor–related genes in HCC, we first searched for genes with decreased expression in HCC samples compared with corresponding normal tissue
We confirmed reduced expression of doublecortin domain-containing 2 (DCDC2) mRNA in tumor tissue by semi-quantitative Reverse transcription-polymerase chain reaction (RT-PCR) in the case whose samples were used for the array analysis (Figure 1a)

Summary

Introduction

To detect genes correlated with hepatocellular carcinoma (HCC), we developed a triple combination array consisting of methylation array, gene expression array and single nucleotide polymorphism (SNP) array analysis. We developed a double combination array analysis consisting of gene expression array and SNP array analysis, and reported a number of tumor suppressor genes in HCC [12,13,14,15,16,17]. In these studies, we hypothesized that DNA methylation of the promoter region of these genes downregulated gene expression, causing HCC progression. We named it triple combination array analysis; this method seems to be an efficient procedure for the detection of tumor suppressor genes of HCC [18]

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