5-氮杂-2'-脱氧胞苷对肺癌SPC-A-1细胞RAR-β基因去甲基化及其表达的作用
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摘要
目的观察5-氮杂-2'-脱氧胞苷(5-Aza-CdR)对肺腺癌SPC-A-1细胞视黄酸受体β(RAR-β)基因甲基化状态的影响,探讨不同浓度5-Aza-CdR对RAR-β基因表达缺陷的调控机制。方法将体外培养的人肺腺癌SPC-A-1细胞株分为4组,分别是A组(未加药)及B、C、D组(分别加入3、6、12μmol/L的5-Aza-CdR)。采用甲基化特异性PCR(MSP)方法检测4组细胞RAR-β基因甲基化状态的差异,RT-PCR法检测4组细胞RAR-βmRNA表达水平的差异,Western blot检测4组细胞RAR-β蛋白表达水平的差异,流式细胞仪技术检测各组细胞周期及细胞凋亡率的差异。结果 A组标本检测出RAR-β基因为甲基化状态,而B、C、D组均检测出RAR-β为去甲基化状态。肺腺癌SPC-A-1细胞RAR-βmRNA及RAR-β蛋白表达水平低下,经5-Aza-CdR处理后,其表达水平明显增强(P<0.01)。5-Aza-CdR处理后(B、C、D组)的细胞凋亡率均较A组明显提高,差异有统计学意义(P<0.01)。结论肺癌RAR-β基因因甲基化出现表达缺陷,5-Aza-CdR具有明显去甲基化作用,可诱导RAR-β基因重新激活表达,促进肺癌细胞凋亡及抑制癌细胞增殖,从而发挥抗癌作用。
Objective To observe effects of 5- Aza- CdR on RAR- β gene methylation status changes in lung adenocarcinoma cells SPC- A- 1,and to investigate the regulatory mechanism of 5- Aza- CdR in different concentrations for defective expression of RAR- β gene. Methods The human lung adenocarcinoma SPC- A- 1 cells were divided into four groups: Group A( control group) and Group B,C,D( dosing groups). Differences in RAR- β gene methylation status,mRNA expression and protein expression among the four groups were assessed by methylation- specific PCR( MSP),RT- PCR,and Western blot,respectively. Furthermore,differences in cell cycle and apoptosis rate among the four groups were detected by Flow cytometry. Results RAR- β gene methylation was observed in Group A,while demethylation of RAR- β gene was observed in the other 3 groups. After 5- Aza- CdR treatment,the expression levels of RAR- β in SPC- A- 1 cells were significantly enhanced( P < 0. 01). Apoptosis rates in 5- Aza- CdR treatment groups were significantly increased compared with Group A( P < 0. 01). Conclusion RAR- β gene in lung cancer is defective expressed due to its methylation,which could be reversed by 5- Aza- CdR via its demethylation effect. 5- Aza-CdR promotes apoptosis and inhibits cell proliferation in lung cancer,and thus plays a role in cancer treatment.
Objective To observe effects of 5- Aza- CdR on RAR- β gene methylation status changes in lung adenocarcinoma cells SPC- A- 1,and to investigate the regulatory mechanism of 5- Aza- CdR in different concentrations for defective expression of RAR- β gene. Methods The human lung adenocarcinoma SPC- A- 1 cells were divided into four groups: Group A( control group) and Group B,C,D( dosing groups). Differences in RAR- β gene methylation status,mRNA expression and protein expression among the four groups were assessed by methylation- specific PCR( MSP),RT- PCR,and Western blot,respectively. Furthermore,differences in cell cycle and apoptosis rate among the four groups were detected by Flow cytometry. Results RAR- β gene methylation was observed in Group A,while demethylation of RAR- β gene was observed in the other 3 groups. After 5- Aza- CdR treatment,the expression levels of RAR- β in SPC- A- 1 cells were significantly enhanced( P < 0. 01). Apoptosis rates in 5- Aza- CdR treatment groups were significantly increased compared with Group A( P < 0. 01). Conclusion RAR- β gene in lung cancer is defective expressed due to its methylation,which could be reversed by 5- Aza- CdR via its demethylation effect. 5- Aza-CdR promotes apoptosis and inhibits cell proliferation in lung cancer,and thus plays a role in cancer treatment.
引文
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[15]Xiang L,Wang R,Wei J,et al.Retinoic acid receptor-βgene reexpression and biological activity in SHI-1 cells after combined treatment with 5-aza-2'-deoxycytidine and all-trans retinoic acid[J].Acta Haematol,2015,133(3):279-286.
[2]Fujii S,Srivastava V,Hegde A,et al.Regulation of AURKC expression by Cp G island methylation in human cancer cells[J].Tumour Biol,2015,36(10):8147-8158.
[3]Heller G,Babinsky VN,Ziegler B,et al.Genome-wide Cp G island methylation analyses in non-small cell lung cancer patients[J].Carcinogenesis,2013,34(3):513-521.
[4]Li KK,Li F,Li QS,et al.DNA methylation as a target of epigenetic therapeutics in cancer[J].Anticancer Agents Med Chem,2013,13(2):242-247.
[5]Moison C,Senamaud-Beaufort C,Fourrière L,et al.DNA methylation associated with polycomb repression in retinoic acid receptorβsilencing[J].FASEB J,2013,27(4):1468-1478.
[6]Brabender J,Metzger R,Salonga D,et al.Comprehensive expression analysis of retinoic acid receptors and retinoid X receptors in non-small cell lung cancer:implications for tumor development and prognosis[J].Carcinogenesis,2005,26(3):525-530.
[7]Füller M,Klein M,Schmidt E,et al.5-azacytidine enhances efficacy of multiple chemotherapy drugs in AML and lung cancer with modulation of Cp G methylation[J].Int J Oncol,2015,46(3):1192-1204.
[8]Pan ZY,Jiang ZS,Ouyang HQ.Study of the methylation patterns of the EGFR gene promoter in non-small cell lung cancer[J].Genet Mol Res,2015,14(3):9813-20
[9]Dong YQ,Liang JS,Zhu SB,et al.Effect of 5-aza-2'-deoxycytidine on cell proliferation of non-small cell lung cancer cell line A549 cells and expression of the TFPI-2 gene[J].Asian Pac J Cancer Prev,2013,14(7):4421-4426.
[10]VispéS,Deroide A,Davoine E,et al.Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2'-deoxycytidine in human leukemic KG1 cells[J].Oncotarget,2015,6(17):15265-15282.
[11]Ines FM,Valeria GG,Lis SM,et al.Retinoic acid reduces migration of human breast cancer cells:role of retinoic acid receptor beta[J].J Cell Mol Med,2014,18(6):1113-1123.
[12]Bhagat R,Kumar SS,Vaderhobli S,et al.Epigenetic alteration of p16 and retinoic acid receptor beta genes in the development of epithelial ovarian carcinoma[J].Tumour Biol,2014,35(9):9069-9078.
[13]Sun SY,Wan H,Yue P,et al.Evidence that retinoic acid receptor beta induction by retinoids is important for tumor cell growth inhibition[J].J Biol Chem,2000,275(22):17149-17153.
[14]张晨烨,金永堂,徐鹤云,等.p16、DAPK和RARβ基因启动子Cp G岛甲基化与非小细胞肺癌临床特征的关系[J].中华医学遗传学杂志,2011,28(1):23-28.
[15]Xiang L,Wang R,Wei J,et al.Retinoic acid receptor-βgene reexpression and biological activity in SHI-1 cells after combined treatment with 5-aza-2'-deoxycytidine and all-trans retinoic acid[J].Acta Haematol,2015,133(3):279-286.