甲基硝基亚硝基胍对哈萨克族人群正常食管上皮细胞p16和FHIT基因甲基化及其表达的影响

Effects of MNNG on methylation and expression of p16 and FHIT genes in normal esophageal epithelial cells in Kazakhs

目的:探讨N-甲基-N'-硝基-N-亚硝基胍(MNNG)对哈萨克族人群正常食管上皮细胞p16和脆性组氨酸三联体(FHIT)基因甲基化状态及其表达的影响。方法:体外培养哈萨克族人群正常食管上皮细胞,用含MNNG浓度分别为0.75、1.50和3.00μg/mL的培养基培养24 h,同时设立空白对照组。采用甲基化特异性聚合酶链(MSP)法检测p16和FHIT基因甲基化状态,实时荧光定量PCR(real time PCR)、Western blot法分别检测p16和FHIT mRNA及蛋白表达水平。结果:与对照组比较,各浓度MNNG处理组细胞p16和FHIT基因甲基化状态均保持未甲基化状态不变。各组细胞p16 mRNA和蛋白表达水平与对照组比较均升高,差异均具有统计学意义(P<0.05或P<0.01);0.75μg/mL MNNG处理组细胞FHIT mRNA表达水平低于对照组(P<0.01),而3.00μg/mL MNNG处理组细胞FHIT mRNA表达水平高于对照组(P<0.05),且3.00μg/mL MNNG处理组FHIT蛋白表达水平较对照组明显升高,差异有统计学意义(P<0.05)。结论:MNNG可促进哈族人群正常食管上皮细胞p16、FHIT mRNA和蛋白的表达。

OBJECTIVE： To investigate the effects of MNNG on methylation and expressions ofp16 and FHIT genes in normal esophageal epithelial cells in Kazakhs. METHODS：Kazakh people’s normal esophageal epithelial cells were culture in vitro in medium containing MNNG concentrations of 0.00,0.75,1.50,and 3.00 mg/mL for 24 h. Methylation ofp16 andFHIT were analyzed by methylmion specific PCR（MSP）,the mRNA and protein level ofp16 and FHIT were measured by real time PCR （RT-PCR） and Western blot. RESULTS：Compared with the control group,p16 andFHIT gene methylation status did not change in treated groups,but the expressions ofp16 mRNA and protein level in treated groups were significantly higher than the control group（P〈0.05 orP〈0.01）. The expressions ofFHIT mRNA level in 0.75μg/mL group was significantly decreased compared with the control group（P＆lt;0.01）,but in 3.00μg/mL groupFHIT mRNA and protein levels were significantly increased（P〈0.05）.CONCLUSION：At certain concentration, MNNG could increase the expressions ofp16 andFHIT in normal esophageal epithelial cells in Kazakhs.