Methylation profiles of thirty four promoter-CpG islands and concordant methylation behaviours of sixteen genes that may contribute to carcinogenesis of astrocytoma

Abstract

Background: Astrocytoma is a common aggressive intracranial tumor and presents a formidable challenge in the clinic. Association of altered DNA methylation patterns of the promoter CpG islands with the expression profile of cancer-related genes, has been found in many human tumors. Therefore, DNA methylation status as such may serve as an epigenetic biomarker for both diagnosis and prognosis of human tumors, including astrocytoma.

Methods: We used the methylation specific PCR in conjunction with sequencing verification to establish the methylation profile of the promoter CpG island of thirty four genes in astrocytoma tissues from fifty three patients (The WHO grading: I: 14, II: 15, III: 12 and IV: 12 cases, respectively). In addition, compatible tissues (normal tissues distant from lesion) from three non-astrocytoma patients were included as the control.

Results: Seventeen genes (ABL, APC, APAF1, BRCA1, CSPG2, DAPK1, hMLH1, LKB1, PTEN, p14ARF, p15INK4b, p27KIP1, p57KIP2, RASSF1C, RB1, SURVIVIN, and VHL) displayed a uniformly unmethylated pattern in all the astrocytoma and non-astrocytoma tissues examined. However, the MAGEA1 gene that was inactivated and hypermethylated in non-astrocytoma tissues, was partially demethylated in 24.5% of the astrocytoma tissues (co-existence of the hypermethylated and demethylated alleles). Of the astrocytoma associated hypermethylated genes, the methylation pattern of the CDH13, cyclin a1, DBCCR1, EPO, MYOD1, and p16INK4a genes changed in no more than 5.66% (3/53) of astrocytoma tissues compared to non-astrocytoma controls, while the RASSF1A, p73, AR, MGMT, CDH1, OCT6, MT1A, WT1, and IRF7 genes were more frequently hypermethylated in 69.8%, 47.2%, 41.5%, 35.8%, 32%, 30.2%, 30.2%, 30.2% and 26.4% of astrocytoma tissues, respectively. Demethylation mediated inducible expression of the CDH13, MAGEA1, MGMT, p73 and RASSF1A genes was established in an astrocytoma cell line (U251), demonstrating that expression of these genes is likely regulated by DNA methylation. AR gene hypermethylation was found exclusively in female patients (22/27, 81%, 0/26, 0%, P < 0.001), while the IRF7 gene hypermethylation preferentially occurred in the male counterparts (11/26, 42.3% to 3/27, 11%, P < 0.05). Applying the mathematic method "the Discovery of Association Rules", we have identified groups consisting of up to three genes that more likely display the altered methylation patterns in concert in astrocytoma.

Conclusions: Of the thirty four genes examined, sixteen genes exhibited astrocytoma associated changes in the methylation profile. In addition to the possible pathological significance, the established concordant methylation profiles of the subsets consisting of two to three target genes may provide useful clues to the development of the useful prognostic as well as diagnostic assays for astrocytoma.

Figure 1

MSP/sequencing analyses of the p16^INK4a gene in astrocytoma and hepatocellular carcinoma Both electrophoretic patterns of the PCR products of the p16^INK4a in each of five astrocytoma cases (21, 22, 26, A11 and B6) and one HCC case (Z92K) (indicated respectively, at the top of figures) were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence.

Figure 2

The methylation state and expression profiles of the CDH13, p73, MAGEA1, MGMT and RASSF1A genes in U251 astrocytoma cells before and after the demethylation treatment with 5-Aza-2'-deoxycytidine U251 cells were subjected to the 10 and 20 nM 5-Aza-2'-deoxycytidine (5-Aza) treatment for 3 days before both DNA and RNA were prepared for either MSP analyses or RT-PCR assessments. Panels; A, the methylation status of the CDH13, p73, MAGEA1, MGMT and RASSF1A genes and B, the expression profiles of each of these five genes, respectively in U251 cells.