Re-expression of RASSF1A by 5-Aza-CdR induced demethylation of the promoter region in human biliary tract carcinoma cells

Abstract

Hypermethylation of the promoter region is an important mean for the transoriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferase are considered to be the main cause of promoter hypermethylation. In order to further explore the epigenetic mechanism of tumor suppressor gene RASSF1A inactivation, 5-aza-2′-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, was used to treat the human biliary tract carcinoma cell line QBC-939 at the concentration of 5 μmol/L for 24 h in this study. After the chemical intervention with 5-Aza-CdR, the methylation status in the promoter region of RASSF1A gene was detected by methylation specific PCR (MS-PCR), and the expression alteration of RASSF1A mRNA and protein were observed by RT-PCR and Western Blot respectively. Following the treatment with 5-Aza-CdR, methylation status in the promoter region of RASSF1A gene was reversed from methylation to unmethylation. A 280 bp DNA band which represented RASS1FA expression at transcriptional level and a 40 kDa (1kDa=0.9921 ku) protein band which represented RASSF1A expression at protein level were detected by RT-PCR and Western Blot respectively in the experimental group cells and there were no corresponding bands in the control group cells. The experimental results suggest that 5-Aza-CdR can induce demethylation in the promoter region of RASSF1A. It can also reverse epigenetic transcriptional silencing caused by DNA methylation and induce the re-expression of RASSF1A in QBC-939. This study also suggest that the mechanism of RASSF1A inactivation is very closely related to the methylation of the promoter region, which may provide a new epigenetic understanding for tumor related gene inactivation and the pathogenesis of biliary tract carcinoma.

Keywords

5-aza-2′-deoxycytidine / RASS1A gene / biliary tract carcinoma / DNA methylation / gene expressio

References

[1] SzyfM.. DNA methylation and demetylation as targets for anticancer therapy. Biochemistry (Mosc), 2005, 70(5): 533-549

[2] ItoM., ItoG., KondoM., et al.. Frequent inactivation of RASSF1A, BLU, and SEMA3B on 3p21.3 by promoter hypermethylation and allele loss in non-small cell lung cancer. Cancer Lett, 2005, 225(1): 131-139

[3] YangB., HouseM. G., GuoM., et al.. Promoter methylation profiles of tumor suppressor genes in intrahepatic and extrahepatic cholangiocarcinoma. Mod Pathol, 2005, 18(3): 412-420

[4] MizunoS., ChijiwaT., OkamuraT., et al.. Expression of DNA methyltransferases DNMT1, 3A, and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia. Blood, 2001, 97(5): 1172-1179

[5] RobertsonK. D., UzvolgyiE., LiangG., et al.. The human DNA methyltransferases (DNMTs) 1, 3a and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors. Nucleic Acids Res, 1999, 27(11): 2291-2298

[6] HermannA., GowherH., JeltschA.. Biochemistry and biology of mammalian DNA methyltransferases. Cell Mol Life Sci, 2004, 61(19–20): 2571-2587

[7] FangJ. Y., YangL., ZhuH. Y., et al.. 5-Aza-2′-deoxycitydine induces demethylation and up-regulates transcription of p16INK4A gene in human gastric cancer cell lines. Chin Med J (Engl), 2004, 117(1): 99-103

[8] DaskalakisM., NguyenT. T., NguyenC., et al.. Demethylation of a hypermethylated P15/INK4B gene in patients with myelodysplastic syndrome by 5-Aza-2′-deoxycytidine (decitabine) treatment. Blood, 2002, 100(8): 2957-2964

[9] KaminskiR., KozarK., NiderlaJ., et al.. Demethylating agent 5-aza-2′-deoxycytidine enhances expression of TNFRI and promotes TNF-mediated apoptosis in vitro and in vivo. Oncol Rep, 2004, 12(3): 509-516

[10] DammannR., LiC., YoonJ. H., et al.. Epeigenetic inactivation of a ras association domain family protein from the lung tumor suppressor locus 3p21.3. Nat Genet, 2000, 25(3): 315-319

[11] ChenY. J., TangQ. B., ZouS. Q.. Inactivation of RASSF1A, the tumor suppressor gene at 3p21.3 in extrahepatic cholangiocarcinoma. World J Gastroenterol, 2005, 11(9): 1333-1338

[12] ChenY. J., TangQ. B., ZouS. Q.. Methylation status of RASSF1A gene promoter in sporadic extrahepatic cholangiocarcinoma. Chin J Exp Surg, 2004, 21(12): 1430-1432

[13] TangQ. B., SunH. W., ZouS. Q.. 5-aza-2-deoxycytidine inhibits the growth of bile duct cancer cell line in vivo and in vitro. Chin J Gen Surg, 2004, 19(5): 295-297

[14] TangQ. B., SunH. W., ZouS. Q.. Effects of 5-aza-2-deoxycytidine on the growth and apoptosis of bile duct cancer cell line. J Huazhong Univ Sci Tech (Health Sci), 2004, 33(1): 34-36

[15] HermanJ. G., GraffJ. R., MyohanenS., et al.. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA, 1996, 93(18): 9821-9826

[16] BurbeeD. G., ForgacsE., Zochbauer-MullerS., et al.. Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression. J Natl Cancer Inst, 2001, 93(9): 691-699

[17] SchagdarsurenginU., WilkensL., SteinemannD., et al.. Frequent epigenetic inactivation of the RASSFIA gene in hepatocellular carcinoma. Oncogene, 2003, 22(12): 1866-1871