5-aza-dC、TSA联合MeCP2的RNA干扰质粒对人肺腺癌细胞株A549生物学行为的影响
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摘要
目的:探讨DNA甲基化转移酶抑制剂5-氮-2'-脱氧胞苷(5-aza-dC)、组蛋白去乙酰化酶抑制剂曲古抑菌素A(TSA)和甲基化结合蛋白MeCP2的RNA干扰载体Psilencer-2.1-U6- MeCP2对人肺腺癌细胞株A549生物学行为的影响。
方法:人肺腺癌细胞株A549细胞用含10%胎牛血清的DMEM培养液培养。利用阳离子脂质体将Psilencer-2.1-U6- MeCP2重组质粒(由本课题组成员2007年构建鉴定,且在mRNA水平和蛋白水平证明可以明显降低MeCP2蛋白的表达)转染到A549细胞中。试验分八组:(1)对照组:不给任何处理的A549细胞;(2)实验组1:A549细胞接种24h后,加入5μmol/L的5-aza-dC;(3)实验组2:A549细胞接种24h后,加入300nmol/L TSA;(4)实验组3:经稳定转染的A549细胞;(5)实验组4:A549细胞接种24h后,加入5μmol/L的5-aza-dC和300nmol/L TSA;(6)实验组5:稳定转染的A549细胞接种24h后,加入5μmol/L的5-aza-dC;(7)实验组6:稳定转染的A549细胞接种24h后,加入300nmol/L TSA;(8)实验组7:稳定转染的A549细胞接种24h后,加入5μmol/L的5-aza-dC和300nmol/L TSA。Annexin V/PI双染法测定各组细胞培养72 h后的凋亡情况;RT-PCR检测各组细胞培养72h后C/EBPαmRNA的表达。
结果:(1)Psilencer-2.1-U6- MeCP2重组质粒能够稳定转染到A549细胞中;(2)5-aza-dC、TSA和Psilencer-2.1-U6- MeCP2重组质粒能够诱导A549细胞凋亡,且两两联合处理组A549细胞凋亡率明显高于单处理组(均P <0.01)、三者联合处理组A549细胞凋亡率高于两两联合处理(均P <0.05),差别有统计学意义;(3)5-aza-dC、TSA和Psilencer-2.1-U6- MeCP2重组质粒能够上调C/EBPα基因mRNA的表达,且两两联合处理组A549细胞中C/EBPαmRNA的表达水平高于单处理组(均P <0.01)、三者联合处理组A549细胞中C/EBPαmRNA的表达水平高于两两联合处理(均P <0.05),差别有统计学意义;(4)细胞C/EBPαmRNA的相对表达量及细胞凋亡率在试验组与对照组组间比较,差异均有统计学意义(P <0.01);(5)5-aza-dC、TSA和Psilencer-2.1-U6- MeCP2重组质粒在诱导细胞凋亡、增强C/EBPαmRNA的表达方面比较,差别均无统计学意义。
结论:与单用5-aza-dC、TSA、Psilencer-2.1-U6- MeCP2重组质粒相比,联合运用能够更好的增强C/EBPα基因mRNA的表达,诱导人肺腺癌A549细胞凋亡,且三者联合运用的效果最显著,这将为去甲基化多药联合治疗肺癌提供理论依据。
AIM:To explore the effects of methyltransferase inhibitor 5-aza-2-deoxycytidine (5-aza-dC), histone deacetylase inhibitor trichostatinA (TSA) and the RNA interference plasmid Psilencer-2.1-U6-MeCP2 of methyl-binding protein MeCP2 on biological behavior in lung epithelial carcinoma A549 cells .
Methods:Human epithelial carcinoma A549 cells were cultured in DMEM culture medium containing 10% Fetal bovine serum. The recombinant plasmid of Psilencer-2.1-U6-MeCP2 (The plasmid was builded and identitied by the subject members in 2007, in the mRNA level and protein level which can significantly reduce the expression of MeCP2 protein) were transfected into A549 cells with LipofectamilleTM 2000. The trial consisted of eight groups: (1) control group: do not give any treatment of the A549 cells;(2) experimental group 1: 24h after inoculation, A549 cells were treated with 5μmol/L 5-aza-dC;(3) experimental group 2 : 24h after inoculation, A549 cells were treated with 300nmol/L TSA;(4) experimental group 3: the transfected A549 cells ;(5) experimental group 4: 24h after inoculation, A549 cells were treated with 5μmol/L 5-aza - dC and 300nmol/L TSA;(6) experimental group 5: 24h after inoculation, the transfected A549 cells were treated with 5μmol/L of 5-aza-dC;(7) experimental group 6: 24h after inoculation, the transfected A549 cells were treated with 300nmol/L TSA;(8) experimental group 7: 24h after inoculation, the transfected A549 cells were treated with 5μmol/L 5-aza - dC and 300nmol/L TSA.The effect of apoptosis in different groups for 72 h was detected by Annexin V / PI double staining. the expression of C/EBPαmRNA in different groups for 72 h was detected by RT-PCR.
Results: (1) The recombinant plasmid of Psilencer-2.1-U6-MeCP2 cloud be stably transfect into A549 cells.(2) 5-aza-dC,TSA and the recombinant plasmid of Psilencer-2.1-U6-MeCP2 can induce A549 cells apoptosis, the apoptosis of A549 cell in the groups of combination with two treatments were significantly higher than single- treatment groups (all P <0.01),the apoptosis of A549 cells in the three combined treatments group was higher than the combination with two treatments groups (all P <0.05), The differences were statistically significant.(3)5-aza-dC,TSA and the recombinant plasmid of Psilencer-2.1-U6-MeCP2 can increase the expression levels of C/EBPαmRNA,the expression levels of C/EBPαmRNA in the groups of combination with two treatments were significantly higher than single- treatment groups (all P <0.01),in the three combined treatments group was higher than the combination of the two treatments groups (all P <0.05),The differences were statistically significant.(4)There were significant differences between experimental groups and control group in relative expression of C/EBPαmRNA and cell apoptosis rate(all P < 0.05).(5)It was no significantly differences between 5-aza-dC group ,the TSA group and the recombinant plasmid of Psilencer-2.1-U6-MeCP2group in induce apoptosis and enhance the expression of C/EBPαmRNA.
Conclusion: Compared with 5-aza-dC, TSA or the recombinant plasmid of Psilencer-2.1-U6-MeCP2 alone, combination treatments can significantly induced apoptosis and enhance C/EBPαmRNA expression,and the effects were most significant in three combined treatment, which will provide a theoretical basis for the demethylation of multi-drug combination therapy of lung cancer.
方法:人肺腺癌细胞株A549细胞用含10%胎牛血清的DMEM培养液培养。利用阳离子脂质体将Psilencer-2.1-U6- MeCP2重组质粒(由本课题组成员2007年构建鉴定,且在mRNA水平和蛋白水平证明可以明显降低MeCP2蛋白的表达)转染到A549细胞中。试验分八组:(1)对照组:不给任何处理的A549细胞;(2)实验组1:A549细胞接种24h后,加入5μmol/L的5-aza-dC;(3)实验组2:A549细胞接种24h后,加入300nmol/L TSA;(4)实验组3:经稳定转染的A549细胞;(5)实验组4:A549细胞接种24h后,加入5μmol/L的5-aza-dC和300nmol/L TSA;(6)实验组5:稳定转染的A549细胞接种24h后,加入5μmol/L的5-aza-dC;(7)实验组6:稳定转染的A549细胞接种24h后,加入300nmol/L TSA;(8)实验组7:稳定转染的A549细胞接种24h后,加入5μmol/L的5-aza-dC和300nmol/L TSA。Annexin V/PI双染法测定各组细胞培养72 h后的凋亡情况;RT-PCR检测各组细胞培养72h后C/EBPαmRNA的表达。
结果:(1)Psilencer-2.1-U6- MeCP2重组质粒能够稳定转染到A549细胞中;(2)5-aza-dC、TSA和Psilencer-2.1-U6- MeCP2重组质粒能够诱导A549细胞凋亡,且两两联合处理组A549细胞凋亡率明显高于单处理组(均P <0.01)、三者联合处理组A549细胞凋亡率高于两两联合处理(均P <0.05),差别有统计学意义;(3)5-aza-dC、TSA和Psilencer-2.1-U6- MeCP2重组质粒能够上调C/EBPα基因mRNA的表达,且两两联合处理组A549细胞中C/EBPαmRNA的表达水平高于单处理组(均P <0.01)、三者联合处理组A549细胞中C/EBPαmRNA的表达水平高于两两联合处理(均P <0.05),差别有统计学意义;(4)细胞C/EBPαmRNA的相对表达量及细胞凋亡率在试验组与对照组组间比较,差异均有统计学意义(P <0.01);(5)5-aza-dC、TSA和Psilencer-2.1-U6- MeCP2重组质粒在诱导细胞凋亡、增强C/EBPαmRNA的表达方面比较,差别均无统计学意义。
结论:与单用5-aza-dC、TSA、Psilencer-2.1-U6- MeCP2重组质粒相比,联合运用能够更好的增强C/EBPα基因mRNA的表达,诱导人肺腺癌A549细胞凋亡,且三者联合运用的效果最显著,这将为去甲基化多药联合治疗肺癌提供理论依据。
AIM:To explore the effects of methyltransferase inhibitor 5-aza-2-deoxycytidine (5-aza-dC), histone deacetylase inhibitor trichostatinA (TSA) and the RNA interference plasmid Psilencer-2.1-U6-MeCP2 of methyl-binding protein MeCP2 on biological behavior in lung epithelial carcinoma A549 cells .
Methods:Human epithelial carcinoma A549 cells were cultured in DMEM culture medium containing 10% Fetal bovine serum. The recombinant plasmid of Psilencer-2.1-U6-MeCP2 (The plasmid was builded and identitied by the subject members in 2007, in the mRNA level and protein level which can significantly reduce the expression of MeCP2 protein) were transfected into A549 cells with LipofectamilleTM 2000. The trial consisted of eight groups: (1) control group: do not give any treatment of the A549 cells;(2) experimental group 1: 24h after inoculation, A549 cells were treated with 5μmol/L 5-aza-dC;(3) experimental group 2 : 24h after inoculation, A549 cells were treated with 300nmol/L TSA;(4) experimental group 3: the transfected A549 cells ;(5) experimental group 4: 24h after inoculation, A549 cells were treated with 5μmol/L 5-aza - dC and 300nmol/L TSA;(6) experimental group 5: 24h after inoculation, the transfected A549 cells were treated with 5μmol/L of 5-aza-dC;(7) experimental group 6: 24h after inoculation, the transfected A549 cells were treated with 300nmol/L TSA;(8) experimental group 7: 24h after inoculation, the transfected A549 cells were treated with 5μmol/L 5-aza - dC and 300nmol/L TSA.The effect of apoptosis in different groups for 72 h was detected by Annexin V / PI double staining. the expression of C/EBPαmRNA in different groups for 72 h was detected by RT-PCR.
Results: (1) The recombinant plasmid of Psilencer-2.1-U6-MeCP2 cloud be stably transfect into A549 cells.(2) 5-aza-dC,TSA and the recombinant plasmid of Psilencer-2.1-U6-MeCP2 can induce A549 cells apoptosis, the apoptosis of A549 cell in the groups of combination with two treatments were significantly higher than single- treatment groups (all P <0.01),the apoptosis of A549 cells in the three combined treatments group was higher than the combination with two treatments groups (all P <0.05), The differences were statistically significant.(3)5-aza-dC,TSA and the recombinant plasmid of Psilencer-2.1-U6-MeCP2 can increase the expression levels of C/EBPαmRNA,the expression levels of C/EBPαmRNA in the groups of combination with two treatments were significantly higher than single- treatment groups (all P <0.01),in the three combined treatments group was higher than the combination of the two treatments groups (all P <0.05),The differences were statistically significant.(4)There were significant differences between experimental groups and control group in relative expression of C/EBPαmRNA and cell apoptosis rate(all P < 0.05).(5)It was no significantly differences between 5-aza-dC group ,the TSA group and the recombinant plasmid of Psilencer-2.1-U6-MeCP2group in induce apoptosis and enhance the expression of C/EBPαmRNA.
Conclusion: Compared with 5-aza-dC, TSA or the recombinant plasmid of Psilencer-2.1-U6-MeCP2 alone, combination treatments can significantly induced apoptosis and enhance C/EBPαmRNA expression,and the effects were most significant in three combined treatment, which will provide a theoretical basis for the demethylation of multi-drug combination therapy of lung cancer.
引文
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[7] Zhu WG,The interaction of histone deacetylase inhibitors and DNA methyltransferase inhibitors in the treatment of human cancer cells.Curr Med Chem Anticancer Aqents 2003,3(3):187-199.
[8] Barault L,Charon-Barra C,Jooste V,et a1.Hypermethylator phenotype in sporadic colon cancer:study on a population-based series of 582 cases.Cancer Res,2008, 168(20):8541-8546.
[9] Adrian Bird.DNA methylation patterns and epigenetic memory.Genes,2002,16(1):6-21.
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[13] Eckel.Antitumor effects of a combined 5-aza-2'deoxycytidine and valproic acid treatment on rhabdomyosarcoma and medulloblastoma in Ptch mutant mice.Cancer Res,2009,69(3): 887-95.
[14]尹慧君,顾红军,徐岷等.5-氮-2′-脱氧胞苷对人肺癌A549细胞株增殖和凋亡的影响.第二军医大学学报,2009,30(5):588-590.
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[14] Fang JY,Yang L,Zhu HY,et al.5-aza-2-deoxycitydine induces demethylation and up-regulates transcription of P16INK4A gene in human gastric cancer cell lines.[J]Chin Med J,2004, 117(1):99-103.
[15] Cantor JP,Iliopoulos D,Rao AS,et al.Epigengtic modulation of endogenous tumor suppressor expression in lung cancer xenografts suppresses tumorigenicity[J]. Int J Cancer,2007, 120(1):24-31.
[16] Tang M, Xu W,Wamg Q, et al.Potential of DNMT and its Epigenetic Regulation for Lung Cancer Therapy.Curr Genomics.2009,10(5):336-352.
[17]姜淑娟,尚建强,张蒿等. TSA对肺腺癌细胞株A549细胞凋亡的影响.肿瘤,2006, 26(11):1007-1010.
[18] Wang CT,Meng M.Experimental study on the effect of trichostatin A on differentiation of human lung carcinoma cell.Zhonghua Yi Xue Za Zhi,2007,87(13):927-929.
[19] Platta CS,Greenblatt DY,Kunnimalaiyaan M,et al.The HDAC inhibitor trichostatin A inhibits growth of small cell lung cancer cells.J Surg Res, 2007,142(2):219-226.
[20] Paul MC,Veronica B,Moshe S.Methylated DNA-bindingprotein2 antisense inhibitors suppressor tumourigenesis of human cancer cell lines in vitro and in vivo. [J]. Carcinogenesis. 2004,25(4):499-507.
[21] Yasuhiro T,Romulo MB,Bjorn H,et al.Epigenetic Modulation of Tumor Suppressor CCAAT/Enhancer Binding ProteinαActivity in Lung Cancer. Journal of the National Cancer Institute,2006,(98):396-406.
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[2]陆再英主编,内科学.第七版.北京:人民卫生出版社,2009:123-134.
[3] Momparler R L.Cancer epigenetics [J].Oncogene,2003,22(42):6479-6483.
[4] Miremadi A,Oestergaard M Z,Pharoah P D,et a1.Cancer gentics of epigenetic genes [J]. Hum Mol Genet,2007,16(1):R28-R49.
[5] Bird A,DNA methylation patters and epigeneticmemory.[J] Genes Dev,2002,16(1):6-21.
[6] Boland CR,Shin SK,Goel A.promoter methylation in the genesis of gastrointestinal cancer.Yonsei Med [J],200950(3):309-321.
[7] Zhu WG,The interaction of histone deacetylase inhibitors and DNA methyltransferase inhibitors in the treatment of human cancer cells.Curr Med Chem Anticancer Aqents 2003,3(3):187-199.
[8] Barault L,Charon-Barra C,Jooste V,et a1.Hypermethylator phenotype in sporadic colon cancer:study on a population-based series of 582 cases.Cancer Res,2008, 168(20):8541-8546.
[9] Adrian Bird.DNA methylation patterns and epigenetic memory.Genes,2002,16(1):6-21.
[10] Peter A J,Daiya T.The role of DNA methylation in mammalian epigenetics.Science,2001, 293(5532):1068-1070.
[11] Ghoshal K,Bai S.DNA methyltransferases as targets for cancer theraph.Drugs Today (Barc), 2007,43(6):395-422.
[12]Oyer JA,Aberrant epigenetic silencing is triggered by a transient reduction in gene expression.PLoS One,2009,4(3):e4832.
[13] Eckel.Antitumor effects of a combined 5-aza-2'deoxycytidine and valproic acid treatment on rhabdomyosarcoma and medulloblastoma in Ptch mutant mice.Cancer Res,2009,69(3): 887-95.
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