Dynamin 3: a new candidate tumor suppressor gene in hepatocellular carcinoma detected by triple combination array analysis

Abstract

Background: To 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.

Methods: Surgical 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.

Results: DNM3 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).

Conclusion: The 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.

Keywords: DNM3; hepatocellular carcinoma; methylation; triple combination array.

Figure 1

Analysis of specimens from a 68-year-old female with HCC. Notes: (A) Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed down-regulation of DNM3 in tumor compared with corresponding normal tissue. (B) Copy number analysis of chromosome 1 using single nucleotide polymorphism arrays in HCC specimens revealed amplification of 1q24.3, containing the DNM3 locus, but no deletions. (C) Methylation specific PCR revealed DNM3 promoter hypermethylation in tumor samples only. Abbreviations: DNM3, dynamin 3; HCC, hepatocellular carcinoma; M, methylated; Ma, marker; N, normal; PCR, polymerase chain reaction; U, unmethylated; T, tumor.

Figure 2

(A) Semi-quantitative RT-PCR revealed reactivation of DNM3 expression in three (Hep3B, HLE, and HuH2) of nine HCC cell lines. (B) MSP revealed complete methylation of DNM3 in HuH2, partial methylation in HepG2, Hep3B, HLE, HLF, HuH1, HuH7, and SK-Hep1 and no methylation in PLC/PRF/5 cells. Abbreviations: HCC, hepatocellular carcinoma; DNM3, dynamin 3; Ma, marker; 5-aza-dC, 5-aza-2′-deoxycytidine; M, methylated; U, unmethylated; MSP, methylation specific PCR. PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction.

Figure 3

Sequence analysis of bisulfite-treated DNA in the DNM3 promoter region. Notes: The methylation status of 31 CpG islands in six clones between −350 and −100 from the transcription initiation site of DNM3 exon 1 is shown. Closed circles represent methylated CpG islands; open circles indicate unmethylated CpG islands. The CpG islands in the DNM3 promoter region in HuH2 cells were abundantly methylated, whereas CpG islands in PLC/PRF/5 cells were predominantly unmethylated. Sequence analysis of CpG islands between −196 and −166 (boxed regions) is shown, where C (black arrows) indicates methylated CpG islands. Bisulfite treatment converts cytosine residues to Ts (green arrows), and indicate unmethylated CpG islands. These results validated the accuracy of MSP and UNMSP in upper region in this figure. Abbreviations: DNA, deoxyribonucleic acid; DNM3, dynamin 3; Ma, marker; 5-aza-dc, 5-aza-2′-deoxycytidine; M, methylated; U, unmethylated; MSP, methylation specific PCR; UNMSP, Unmethylated-Specific PCR; PCR, polymerase chain reaction.

Figure 4

(A) Methylation status of DNM3 in 48 primary HCC samples. Thirty-three of 48 (68.7%) cancer tissues exhibited hypermethylation of DNM3, compared to 13 of 48 (27.0%) cases in adjacent, normal tissues. (B) Four representative cases showing hypermethylation of the promoter region of DNM3 in tumor tissues and no methylation in normal tissues. Abbreviations: HCC, hepatocellular carcinoma; M, methylated; U, unmethylated; DNM3, dynamin 3.

Figure 5

Expression levels of DNM3 mRNA in specimens from 48 patients with HCC. The expression index [(DNM3-tumor) × (GAPDH-normal)/(DNM3-normal) × (GAPDG-tumor)] was calculated for all 48 cases. A trend towards a lower expression index in methylated cases compared to unmethylated cases was observed, however this was not statistically significant (P = 0.189). Abbreviation: HCC, hepatocellular carcinoma; DNM3, dynamin 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 6

Disease specific survival stratified by DNM3 (dynamin 3) expression status. Patients with lower DNM3 expression exhibited poorer prognosis with decreased disease specific survival, compared to patients with higher DNM3 expression (P = 0.014). Note: *, statistically significant.