Aberrant CpG Island Hypermethylation of the RUNX3 Gene in Osteosarcoma

Joo Han Oh; Ilkyu Han; June Hyuk Kim; Gyeong Hoon Kang; Hwan Seong Cho; Seung Cheol Kang; Mi-Ra Lee; Han-Soo Kim

doi:10.4055/jkoa.2009.44.3.285

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Oh, Han, Kim, Kang, Cho, Kang, Lee, and Kim: Aberrant CpG Island Hypermethylation of the RUNX3 Gene in Osteosarcoma

Original Article

Journal of the Korean Orthopaedic Association

Journal of the Korean Orthopaedic Association 2009; 44(3): 285-293.

Published online: 18 June 2009

DOI: https://doi.org/10.4055/jkoa.2009.44.3.285

Aberrant CpG Island Hypermethylation of the RUNX3 Gene in Osteosarcoma

Joo Han Oh, M.D., Ilkyu Han, M.D., June Hyuk Kim, M.D., Gyeong Hoon Kang, M.D.^*, Hwan Seong Cho, M.D.^†, Seung Cheol Kang, M.D., Mi-Ra Lee, M.D., Han-Soo Kim, M.D.

Department of Orthopaedic Surgery, Seoul National University College of Medicine, Seoul, Korea.

^*Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.

^†Department of Orthopedic Surgery, Kyungpook National University School of Medicine, Daegu, Korea.

Address reprint requests to Han-Soo Kim, M.D. Department of Orthopaedic Surgery, Seoul National University College of Medicine, 101 Daehangno, Jongno-gu, Seoul 110-744, Korea. Tel: +82.2-2072-2362, Fax: +82.2-764-2718, hankim@snu.ac.kr

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.

Keywords: RUNX3, Osteosarcoma, Methylation, CpG island

Figures and Tables

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.

Table 1

Primer Sequences and PCR Conditions of Tumor Suppressor Genes for Methylation-specific PCR

^*annealing temperature, ^†M, methylated; ^‡U, unmethylated.

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