Methylation Status Of RASSF1A Promoter And Growth Arrested By Demethylation Of 5-Aza-cdR In Retinoblastoma Cells

Part1 Methylation status of RASSF1A and DAPK promoter in retinoblastomaObjectiveTo investigate the methylation status of RASSF1A and DAPK promoter in retinoblastoma tissue or in peripheral blood of patients with retinoblastoma.MethodsMethylation status of RASSF1A and DAPK promoter in 28 RB tissues and 9 cases peripheral blood of patients with RB were detected by methylation specific PCR(MSP),and the difference of methylation were compared among the different groups.Results1.The percentage of RASSF1A hypermethylation in RB tissues (60.7%)was much higher than in normal retinal tissue and adjacent normal retinal tissue with statistics difference(P＜0.01).2.RASSF1A hypermethylation percentage in tumor tissues of the unilateral RB was higher than of the bilateral(P＜0.05).3.RASSF1A hypermethylation was detected in 2 out of 9 cases of peripheral blood from patients with RB,without statistics difference compared with that of normal person.4.No DAPK promoter hypermethylation was found in all RB tissues and plasma from retinoblastoma patients.ConclusionRASSF1A promoter hypermethylation was frequently present in retinoblastoma. Part 2 Effect of 5-Aza-CdR on RASSF1A promoter demethylation in RB cells at transcriptional levelObjectiveTo study the effects of 5-Aza-CdR on the transcription regulation of promoter demethylation on RASSFIA in HXO-RB₄₄cell line.MethodsHXO-RB₄₄cells were divided into experiment groups randomly, according to different concentration of 5-Aza-CdR:B(0.1μmol/l); C(0.5μmol/l);D(1.0μmol/l);E(5.0μmol/l)and F(10.0μmol/l).And 0.0125%DMSO was used as vehicle control.The methylation status of RASSF1A gene promoter was detected by MSP on the 4th day after incubation.And RASSF1A mRNA expression was analyzed at the concentration of 5.0μmol/l by RT-PCR at 1d,2d,3d,4d,5d,6d,7d, respectively.Immunohistochemical was also used to detect the expression of RASSF1A protein in HXO-RB₄₄cells.Results1.Photodensity(OD)of methylation of RASSF1A gene decreased gradually and that of unmethylation was increased with increasing concentration(0.5μmol/l -5.0μmol/l)of 5-Aza-CdR in HXO-RB₄₄cells up to the peak at 5.0μmol/l with statistics difference compared with any other groups. 2.The silence-expressing RASSF1A mRNA in HXO-RB₄₄cells was re-expressed at 5d,6d,7d,induced by 5.0μmol/l 5-Aza-CdR.Compared with control,RASSF1A mRNA significantly increased on the 5th day (P＜0.05).3.RASSF1A protein was re-expressed in HXO-RB₄₄cells induced by 5.0μmol/l 5-Aza-CdR on 5th day,while negative in control group A.Conclusion1.The expression of RASSF1A mRNA and protein was absent in HXO-RB₄₄cell lines because of the promoter hypermethylation of RASSF1A gene.2.The methylation status was reversed from 0.5μmol/l to 5.0μmol/l and the RASSF1A transcripts were restored after 5.0μmol/l 5-Aza-CdR treated on the 5th day in HXO-RB₄₄cell line.3.RASSF1A promoter hypermethylation maybe a valuable target in the therapy of RB. Part 3 Effect of 5-Aza-CdR on arresting growth of RB cells in vitroObjectiveTo study the effect of 5-Aza-CdR on biological behavior of HXO-RB₄₄cells in vitro and may provide a new treatment strategy for RB.MethodsProtocol of experiment grouping and cells treatment was the same as that in Part 2.Absorbance(A)at different time(24h,48h,72h,96h,120h,144h)induced by different concentration of 5-Aza-CdR was detected by MTT assay,cell cycle and apoptosis assessed by flow cytometry.Results1.Induced by 5.0μmol/l 5-Aza-CdR 5-Aza-CdR,HXO-RB₄₄cells showed decreased proliferation and growth velocity with statistics difference(P＜0.05)2.Cell cycle and apoptosis of HXO-RB₄₄cells were altered after induction of 5.0μmol/l 5-Aza-CdR.Population of cells in G₀/G₁ and apoptosis was both increased,compared with control group.ConclusionProliferation of HXO-RB₄₄cells were arrested by 5-Aza-CdR via blocking cell cycle at phase G₀/G₁.