5-Aza-CdR对人食管鳞癌Kyse-140细胞BNIP3基因甲基化生物学意义的研究

Biological significance of 5-Aza-CdR on methylation of BNIP3 gene in human esophageal carcinoma cell line Kyse-140

目的:研究DNA甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷(5-azacytidine-2′-deoxvcytidines,5-Aza-CdR)对人食管癌(esophageal squamous cell carcinoma,ESCC)Kyse-140细胞系中Bcl-2/腺病毒E1B19×103相关蛋白3(BNIP3)基因启动子区甲基化的转录调控以及对细胞生长、增殖的影响。方法:培养ESCC Kyse-140细胞,用不同浓度的5-Aza-CdR处理细胞后,倒置显微镜下观察细胞形态的变化,MTT比色法检测5-Aza-CdR对Kyse-140细胞增殖能力的影响;应用甲基化特异性PCR(MSP)、反转录聚合酶链(RT-PCR)及蛋白质印迹法分析经5-Aza-CdR处理前后BNIP3基因甲基化状态及mRNA和蛋白表达水平的变化。结果:倒置显微镜下显示,经5-Aza-CdR处理后,可见细胞增殖速度减慢,体积变小,形态趋于规则,密度降低,细胞间接触变松,核固缩,核分裂像减少,部分细胞有死亡,随药物浓度的增加该趋势更加明显。MTT检测发现,经1×10-7、5×10-7、2×10-6、1×10-5和5×10-5 mol/L的5-Aza-CdR处理后,24h时Kyse-140细胞生长抑制率分别为6.2%、8.4%、11.3%、15.8%和26.0%,48h时分别为8.1%、15.2%、20.4%、24.6%和39.0%,72h分别为9.3%、20.5%、30.1%、40.9%和51.0%,随作用时间的增长细胞抑制率明显升高。方差分析显示,5-Aza-CdR对Kyse-140细胞生长有抑制作用,呈浓度依赖性(F=18.211,P<0.01)和时间依赖性(F=15.411,P=0.002)。BNIP3基因在Kyse-140细胞中呈高甲基化状态,在mRNA和蛋白水平表达阴性;5-Aza-CdR可逆转BNIP3基因的甲基化状态,恢复BNIP3mRNA和蛋白水平的表达,并随药物浓度的增加表达增高。结论:5-Aza-CdR能够逆转BNIP3基因在ESCC Kyse-140细胞株中高甲基化状态,解除DNA高甲基化导致的基因沉默,从而恢复其mRNA和蛋白水平的表达,同时显著抑制细胞的生长和增殖能力。

OBJECTIVE： To investigate the effects of methyltransferase inhibitor 5-azacytidine-2＇-deoxvcytidines （5-Aza-CdR） on the transcriptional regulation through methylation of Bel-2/adenovirus E1B 19 kd-interacting protein 3 （BNIP3） gene promoter region in human esophageal carcinoma cell line Kyse-140 and its effect on the abilities of growth and proliferation the cell line. METHODS： Human esophageal carcinoma cell line Kyse-140 were cultured and treated with different concentrations of 5-Aza-CdR. Morphological changes of cells were observed under inverted microscope and the proliferative ability of the cell was evaluated by MTT assay. Methylation specific PCR （MSP）, RT-PCR and Western blotting were used to detect CpG islands methylation status,the expression of BNIP3 mRNA and protein in Kyse-140 cell line before and after treating with different concentrations of 5-Aza-CdR. RESULTS： Being treated with 1×10^-7, 5 ×10^-7 ,2×10^-6, 1×10^-5 and 5×10^-5 mol/L 5-Aza-CdR, the inhibition rates of 24 hours were 6.2%,8.4%,11.3%, 15.8 % and 26.0%, the inhibition rates of 48 hours were 8.1%, 15.2 %, 20.4 %, 24.6 % and 39.0%, and the inhibition rates of 72 hours were 9.3 %, 20.5%, 30.1%, 40.9 % and 51.0 %. After treated with 5-Aza-CdR, the inhibit ability of tumorcells proliferation was found, and the trend was concentration-dependent （F= 18.211, P〈0. 001） and time-dependent （F= 15. 411,P=0. 002）. Hypermethylation status of BNIP3 gene promoter region was observed in Kyse-140 cell line and no expression of the mRNA and protein of BNIP3 gene was found. 5-Aza-CdR could reverse BNIP3 gene methylation in Kyse-140 cells and strongly up-regulate the expression of BNIP3 mRNA and protein. CONCLUSIONS.. 5-Aza-CdR can re- verse the methylation status of BNIP3 gene in human esophageal carcionma cell line Kyse-140, which would remove the gene silencing caused by high methylation and thus restore the expression the BNIP3 mRNA and protein levels. It can ef- fectively inhibit growth and proliferation of esophageal cancer of esophageal cancer cells.