Home > Archive>Volume 7, Issue 1, 2014 >51-56. DOI:10.3980/j.issn.2222-3959.2014.01.09
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5-Aza-2’-deoxycytidine inhibits retinoblastoma cell by reactivating epigenetically silenced RASSF1A gene
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Supported by the National Natural Science Foundation of China (No.3087282; No.81072221).
Conflicts of Interest: Liu R, None; Zhang XH, None; Zhang K, None; Li W, None; Wang WJ, None; Luo DX, None; Gao L, None.
ACKNOWLEDGEMENTS: The authors thank Dr. De-Liang Cao in Department of Microbiology, Immunology and Cell Biology, Simmons Cancer Institute, Southern Illinois University School of Medicine for proofreading this article. The authors thank Guang-Xiu Lu for approval of the project, which was undertaken in the Institute of Reproduction and Stem Cell Engineering, and all the researchers in the Institute for technical support.

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    Abstract:

    AIM:To investigate the effect of 5-Aza-2’-deoxycytidine (5-Aza-CdR), a DNA methyltransferase (DNMT) inhibitor, on the growth and survival of the Chinese retinoblastoma (RB) cell line HXO-RB44.METHODS:The DNA methylation status of the Ras association domain family (RASSF1A) promoter in the presence of 5-Aza-CdR at different concentrations was analyzed by methylation-specific polymerase chain reaction (MSP). RASSF1A mRNA and protein levels were measured by semiquantitative RT-PCR and immunohistochemistry staining, respectively, when cells were treated with 5.0μmol/L of 5-Aza-CdR. The effect of 5.0μmol/L 5-Aza-CdR on the proliferation and viability of HXO-RB44 cells was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry.RESULTS:5-Aza-CdR efficiently induced cell cycle arrest at G0/G1 and apoptotic death in HXO-RB44 cells. MSP analysis showed that unmethylated RASSF1A DNA increased and methylated RASSF1A decreased in a dose-dependent manner in a range of 0.5-5.0μmol/L 5-Aza-CdR. Accordingly, RASSF1A expression was reactivated at both mRNA and protein levels. Incubation time of 5-Aza-CdR treatment also functioned as a factor for the demethylation status of RASSF1A promoter DNA, with a plateau on day four. 5-Aza-CdR at 5.0μmol/L completely demethylated the RASSF1A promoter in HXO-RB44 cells on day four, and as a result, RASSF1A expression increased significantly from day 4 to day 7.CONCLUSION: 5-Aza-CdR inhibits the growth of the HXO-RB44 RB cell line and induces apoptosis by demethylating the RASSF1A gene.

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Ru Liu, Xiao-Huan Zhang, Kun Zhang,/et al.5-Aza-2’-deoxycytidine inhibits retinoblastoma cell by reactivating epigenetically silenced RASSF1A gene. Int J Ophthalmol, 2014,7(1):51-56

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Publication History
  • Received:June 19,2013
  • Revised:October 23,2013
  • Adopted:October 23,2013
  • Online: February 20,2014
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