腺苷干预人结直肠癌SW480 细胞RECK 基因甲基化机制研究.docVIP

腺苷干预人结直肠癌SW480细胞RECK基因甲基化机制研究 李 权1，王亚旭1，申海鹰2，谢 凯1，张灵敏1，曾令海1 （400010重庆，重庆医科大学附属第二医院胃肠外科1；97232 美国，美国俄勒冈州Legacy研究所2） 【摘要】目的 探讨腺苷干预人结直肠癌SW480细胞RECK基因甲基化及其机制。方法 分别用0，3.0 mmol/L的腺苷干预SW480细胞72 h后，亚硫酸氢盐测序法(BSP)检测RECK基因启动子区CpG岛甲基化情况；逆转录PCR、Western 印迹法、流式细胞仪检测腺苷处理前后RECK基因mRNA和蛋白的表达情况、甲基转移酶（Methyltransferases DNMT1，DNMT3a）蛋白表达情况及细胞凋亡的变化。结果 腺苷干预后RECK基因甲基化程度为(49.14±2.38)%，明显低于对照组（P0.01)；腺苷干预后RECK基因mRNA转录及蛋白表达水平、细胞凋亡水平均明显高于对照组[0.3353±0.3502 vs 0.0800±0.0036，0.6030±0.0178 vs 0.0900±0.0030，(27.90±2.32)% vs (5.09±0.30)%，均P 0.01]；甲基转移酶（DNMT1、DNMT3a）蛋白表达情况均明显低于对照组（0.1413±0.0021 vs 0.6450±0.0092，0.1300±0.0046 vs 0.5267±0.0095，均P 0.01）。结论 腺苷通过抑制甲基转移酶的活性，降低RECK基因DNA启动子区域的甲基化水平，使RECK基因mRNA及蛋白表达水平增加，促使SW480细胞凋亡。 【关键词】 结直肠肿瘤；RECK基因；腺苷；甲基转移酶；细胞凋亡 【中图法分类号】R969.4；R735.3+5；R735.3+7；R394.3 【文献标志码】 A Effects of adenosine in human colorectal cancer SW480 cells RECK gene methylation and mechanisms are investigated Li Quan1 , Wang Yaxu1, Shen Haiying2, Xie Kai1,Zhang Lingmin1, Zeng Linghai1 （1Department of Gastraintestinal Surgery, the Second Affliated Hospital of Chongqing Medical University, Chongqing 400010, China；2Legacy in Oregon research institute in the United States, the United States 97232） 【Abstract】 Objective The effects of adenosine in human colorectal cancer SW480 cells RECK gene methylation and mechanisms are investigated. Methods The SW480 cells were treated with adenosine at concentrations 0, 3.0mmol/L for 72h, and then the CpG islands methylatio levels of the RECK gene promoter region were detected by Bisulfite sequencing polymerase chain reaction (BSP). The RECK gene mRNA and protein expression, the methyl transferase (DNMT1, DNMT3a) protein expression and the cell apoptosis of the adenosine before and after processing were detected by Rt-PCR and western imprinting method, using the flow cytometry. Results After the intervention of adenosine, the RECK gene methylation levels was 49.14% ± 2.38%, it’s significantly lower than the control group(74.84% ± 2.38%, both P 0.01)