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  

The effects of 5-aza-dC,TSA and the RNA interference plasmid of MeCP2 on biological behavior in lung adenocarcinoma A549 cells

作　　者：董必文[1] 段凤英[1] 

机构地区：[1]南昌大学第二附属医院呼吸内科,江西南昌330006

出　　处：《实验与检验医学》2010年第2期110-113,136,共5页Experimental and Laboratory Medicine

基　　金：江西省科学技术厅社会发展攻关项目(编号:837)

摘　　要：目的探讨DNA甲基化转移酶抑制剂5-氮-2'-脱氧胞(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重组质粒转染到A549细胞中。用5μmol/L的5-aza-dC和(或)300nmol/L的TSA处理A549细胞和稳定转染的A549细胞。Annexin V/PI双染法测定各组细胞培养72h后的凋亡情况;应用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重组质粒均能够上调A549细胞C/EBPα基因mRNA的表达、诱导细胞凋亡,且两两联合处理组A549细胞中C/EBPαmRNA的表达水平及凋亡率均明显高于单处理组(均P<0.01)、细胞C/EBPαmRNA的相对表达量及凋亡率在三者联合处理组与两两联合处理组组间比较,差别均有统计学意义(P<0.05);(3)5-aza-dC组、TSA组和Psilencer-2.1-U6-MeCP2重组质粒组在诱导细胞凋亡、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诱导人肺腺癌A549细胞凋亡,增强C/EBPα基因mRNA的表达,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。Object To explore the effects of methyltransferase inhibitor 5-aza-2-deoxycytidine（5-aza-dC）,histone deacetylase inhibitor trichostatin A（TSA）and RNA interference vector Psilencer-2.1-U6-MeCP2 of methyl-binding protein MeCP2 on biological behavior in lung adenocarcinoma A549 cells.Methods Human lung adenocarcinoma A549 cells were cultured in DMEM culture medium containing 10% Fetal bovine serum.The recombinant plasmid of Psilencer-2.1-U6-MeCP2 was transfected into A549 cells.A549 cells and A549 cells of stable transfection were treated with 5μmol/L of 5-aza-dC and（or）300nmol/L of TSA.In the different groups for 72h,the effect of apoptosis was detected by Annexin V /PI double staining,and the expression of C/EBPα mRNA by RT-PCR.Results（1）The recombinant plasmid of Psilencer-2.1-U6-MeCP2 could be stably transfected into A549 cells.（2）5-aza-dC,TSA and the recombinant plasmid of Psilencer-2.1-U6-MeCP2 were able to increased C/EBPα mRNA expression and induce apoptosis in A549 cells.The apoptosis rate and the ex-pression level of C/EBPα mRNA of A549 cells in the groups of combination with two treatments were signifi-cantly higher than those of single-treatment group（all P0.01）.There were significant differences between com-bination of the two combined treatment group and the three combined treatment groups in relative expression of C/EBPα mRNA and apoptosis rate of A549（all P0.05）.（3）There were no significant differences between above three groups in apoptosis and enhanced the expression of C/EBPα mRNA.Conclusions Compared with 5-aza-dC,TSA or the recombinant plasmid of Psi-lencer-2.1-U6-MeCP2 alone,combination treatment cansignificantly induce apoptosis and enhance C/EBPα mR-NA expression,and the effects show the most significant in three combined treatment,which will provide a theo-retical basis for the demethylation of multi-drug combination therapy.

关 键 词：肺肿瘤 曲古抑菌素A 5-氮-2'-脱氧胞苷 RNA干扰 C/EBPΑ 细胞凋亡 A549细胞 

分 类 号：R734.2[医药卫生—肿瘤] R329.28[医药卫生—临床医学]