Summary
In this study, RNA interference technique was employed to silence the expression of DNMT1 and/or DNMT3b in human bladder cancer T24 cells. The expression levels of their mRNA and protein were greatly decreased by up to 75% and 65% respectively after T24 cells were transfected with lipofectamine2000 for 72 h, indicating RNA interference is an effective tool in gene knockdown. Proliferation and apoptosis of T24 cells were detected by MTT, and annexin-V-FITC and propidium iodide staining flow cytometry, respectively. It was found that loss of the DNMT1 or DNMT3b expression could inhibit the cell growth and promote the cell apoptosis to some extent. However, combined treatment with shRNA targeting both DNMT1 and DNMT3b mRNA could obviously enhance the above effects. It was concluded that simultaneously silencing both genes could result in strong suppressing effect on tumor proliferation and promoting cell apoptosis than separate use, suggesting combined use of DNMT1 and DNMT3b can achieve a synergistic effect in the CpG island methylation in human bladder tumorigenesis.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998,391(6669):806–811
Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001,411(6836):494–498
Waterhouse PM, Wang MB, Lough T. Gene silencing as an adaptive defence against viruses. Nature, 2001,411 (6839):834–842
Szyf M, Detich N. Regulation of the DNA methylation machinery and its role in cellular transformation. Prog Nucleic Acid Res Mol Biol, 2001,69:47–79
Kanai Y, Hirohashi S. Alterations of DNA methylation associated with abnormalities of DNA methyltransferases in human cancers during transition from a precancerous to a malignant state. Carcinogenesis, 2007,28(12):2434–2442
Luczak MW, Jagodzinski PP. The role of DNA methylation in cancer development. Folia Histochem Cytobiol, 2006,44(3):143–154
Bestor TH. The DNA methyltransferases of mammals. Hum Mol Genet, 2000,9(16):2395–2402
Margot JB, Ehrenhofer-Murray AE, Leonhardt H. Interactions within the mammalian DNA methyltransferase family. BMC Mol Biol, 2003,4(7):1471–1479
Wu JC, Santi DV. On the mechanism and inhibition of DNA cytosine methyltransferases. Prog Clin Biol Res, 1985,198:119–1929
Robertson KD, Uzvolgyi E, Liang G, 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
Nakagawa T, Kanai Y, Ushijiama S, et al. DNA hypermethylation on multiple CpG islands associated with increased DNA methyltransferase DNMT1 protein expression during multistage urothelial carcinogenesis. J Urol, 2005,173(5):1767–1771
Etoh T, Kanai Y, Ushijiama S, et al. Increased DNA methyltransferase 1 (DNMT1) protein expression correlates significantly with poorer tumor differentiation and frequent DNA hypermethylation of multiple CpG islands in gastric cancers. Am J Pathol, 2004,164(2):689–699
Chen CL, Yan X, Gao YN, et al. Expression of DNA methyltransferase 1, 3A and 3B mRNA in the epithelial ovarian carcinoma. Zhonghua Fu Chan Ke Za Zhi (Chinese), 2005,40(11):770–774
Benbrahim-Tallaa L, Waterland RA, Dill AL, et al. Tumor suppressor gene inactivation during cadmium-induced malignant transformation of human prostate cells correlates with overexpression of de novo DNA methyltransferase. Environ Health Perspect, 2007,115(10):1454–1459
Fanelli M, Caprodossi S, Ricci-Vitiani L, et al. Loss of pericentromeric DNA methylation pattern in human glioblastoma is associated with altered DNA methyltransferases expression and involves the stem cell compartment. Oncogene, 2008,10:27 (3):358–365
Szyf M. Targeting DNA methylation in cancer. Bull Cancer, 2006,93(9):961–972
Esteller M. Relevance of DNA methylation in the management of cancer. Lancet Oncol, 2003,4(6):351–358
Kassis ES, Zhao M, Hong JA, et al. Depletion of DNA methyltransferase 1 and/or DNA methyltransferase 3b mediates growth arrest and apoptosis in lung and eso-phageal cancer and malignant pleural mesothelioma cells. J Thorac Cardiovasc Surg, 2006,131(2):298–306
Xiong Y, Dowdy SC, Xue A, et al. Opposite alterations of DNA methyltransferase gene expression in endometrioid and serous endometrial cancers. Gynecol Oncol, 2005, 96(3):601–609
Robert MF, Morin S, Beaulieu N, et al. DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells. Nat Genet, 2003,33(1):61–65
Leu YW, Ralunatpanah F, Shi H, et al. Double RNA interference of DNMT3b and DNMT1 enhances DNA de-methylation and gene reactivation. Cancer Res, 2003, 63(19):6110–6115
Rhee I, Jair KW, Yen RW, et al. CpG methylation is maintained in human cancer cells lacking DNMT1. Nature, 2000,404(6781):1003–1007
Author information
Authors and Affiliations
Additional information
The project was supported by a grant from a Science and Technology Research Program of Hubei Province (No. 2006AA301B56-1).
Rights and permissions
About this article
Cite this article
Zhang, S., Zeng, F., Peng, S. et al. Effects on biological behavior of bladder carcinoma T24 cells via silencing DNMT1 and/or DNMT3b with shRNA In Vitro . J. Huazhong Univ. Sci. Technol. [Med. Sci.] 29, 215–219 (2009). https://doi.org/10.1007/s11596-009-0216-z
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11596-009-0216-z