J Korean Orthop Assoc.  2009 Jun;44(3):285-293. 10.4055/jkoa.2009.44.3.285.

Aberrant CpG Island Hypermethylation of the RUNX3 Gene in Osteosarcoma

• Oh JH
• Han I
• Kim JH
• Kang GH
• Cho HS
• Kang SC
• Lee MR
• Kim HS

• KMID: 2186336
• DOI: http://doi.org/10.4055/jkoa.2009.44.3.285

Abstract

PURPOSE: Transcriptional silencing of tumor suppressor genes by aberrant methylation of CpG islands plays a crucial role in the development of human cancers. We comprehensively examined the methylation status of several tumor suppressor genes in osteosarcoma with a special focus on the RUNX3 gene.
MATERIALS AND METHODS
Methylation-specific polymerase chain reaction (MSP) was performed for osteosarcoma tissues and their cell lines. MSP and RT-PCR for the RUNX3 gene were performed in the tumor-derived cell lines and the immortalized cell lines. The demethylating agent 5-aza-2' deoxycytidine was used in the SaOS-2 cell line to reverse the methylation status.
RESULTS
Hypermethylation of the RUNX3 gene was observed in 60% (24 of 40) of the osteosarcoma tissues, whereas other tumor suppressor genes showed very low methylation. Thirteen of 30 (43%) tumor-derived cell lines, and U-2OS and SaOS-2 showed hypermethylation of the RUNX3 gene on MSPCR. However, RUNX3 was expressed in the SaOS-2 cell line, as determined by RT-PCR, and the expression was augmented by treatment with 5-aza-2' deoxycytidine.
CONCLUSION
Our study suggests that aberrant methylation is an important mechanism of RUNX3 down-regulation in osteosarcoma. This data may have potential significance in developing a potential therapeutic target for osteosarcoma.

Keyword

RUNX3; Osteosarcoma; Methylation; CpG island

MeSH Terms

Cell Line
CpG Islands
Deoxycytidine
Down-Regulation
Genes, Tumor Suppressor
Humans
Methylation
Osteosarcoma
Polymerase Chain Reaction
Deoxycytidine

Figure

• 

  Fig. 1 Methylation status of various tumor suppressor genes in osteosarcoma (n=40).

• 

  Fig. 2 Methylation of RUNX3 gene using methylation-specific PCR in osteosarcoma cell lines. (A) The results of 10 representative cell lines cultured from osteosarcoma tissues are shown. Numbers 2, 3, 7, 8 show methylated bands. (B) The results of 4 immortalized cell lines are shown. SaOS-2 and U-2OS showed methylation of RUNX3.

• 

  Fig. 3 Expression of RUNX3 in SaOS-2 cell line by RT-PCR. RUNX3 was expressed in SaOS-2 cell line. Treatment with Trichostatin A (TSA) down-regulated RUNX3 expression, which was reversed by co-treatment with 5-aza-2' deoxycytidine (5-aza-dC). Lane 1: control, lane 2: 5-aza-dC, lane 3: TSA, lane 4: 5-aza-dC+TSA.

• 

  Fig. 4 Bisulfite genomic sequencing of 4 immortalized osteosarcoma cell lines. Most of the CpG islands were methylated in SaOS-2 and U-2OS cell lines. However, some unmethylated CpG sires were also observed.

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