甲基硝基亚硝基胍对哈萨克族正常食管上皮细胞LRRFIP1、ALDH1L1基因甲基化及其表达的影响

Effects of MNNG on methylation and expression of LRRFIP1,ALDH1L1 on Kazakh normal esophageal epithelial cells

目的探讨N-甲基-N'-硝基-N-亚硝基胍(MNNG)对哈萨克族(哈族)正常食管上皮细胞LRRFIP1、ALDH1L1基因甲基化及其表达的影响。方法将体外培养的哈族正常食管上皮细胞暴露于含0、0.75、1.5、3μg/mL MNNG的培养基中24 h,分别为对照组、0.75μg/mL组、1.50μg/mL组、3.00μg/mL组。用甲基化特异性聚合酶链(MSP)法检测LRRFIP1、ALDH1L1基因甲基化状态,用RT-PCR和Western Blot法检测LRRFIP1、ALDH1L1基因mRNA和蛋白表达。结果与对照组比较,各浓度组细胞LRRFIP1基因甲基化状态没有改变;各组细胞LRRFIP1基因mRNA表达水平分别为(1.021±0.237)、(2.828±0.350)、(1.453±0.179)、(1.952±0.223);与对照组比较,0.75μg/mL组和3.00μg/mL组表达均升高,差异均具有统计学意义(P<0.05);与对照组比较,1.50μg/mL和3.00μg/mL组LRRFIP1蛋白表达水平均升高,分别为(4.233±0.048)、(4.519±0.033)、(5.377±0.057),差异有统计学意义(P<0.05);高浓度组细胞ALDH1L1基因有从部分甲基化向完全甲基化转变的趋势,各组ALDH1L1基因mRNA表达水平分别为(1.023±0.254)、(5.046±0.603)、(2.259±0.030)、(7.616±1.910),与对照组比较各浓度组均升高,差异均具有统计学意义(P<0.05);各组细胞ALDH1L1蛋白表达水平分别为(0.725±0.014)、(0.913±0.033)、(1.142±0.010)、(1.334±0.047),与对照组比较各浓度组均升高,差异均具有统计学意义(P<0.05)。结论 MNNG可促进哈族正常食管上皮细胞ALDH1L1基因甲基化及LRRFIP1、ALDH1L1表达的升高。

Objective To investigate the effects of MNNG on methylation and expressions of LRRFIP1 and ALDH1L1 on Kazakh normal esophageal epithelial cells.Methods Methylation of LRRFIP1 and ALDH1L1 were detected by MSP,the mRNA and protein level of LRRFIP1 and ALDH1L1 were detected by RT-PCR and Western Blot.Results Compared with the control group,each group of cells with LRRFIP1 gene methylation status did not change,the expressions of LRRFIP1 mRNA level in each groups were（1.021 ± 0.237）,（2.828 ± 0.350）,（1.453 ± 0.179）,（1.952 ± 0.223）,the LRRFIP1 mRNA level in low concentration group and high concentration group of cells were significantly higher than the control group（P〈0.05）,the expressions of LRRFIP1 protein level in control group and high concentration group were（4.233 ± 0.048）,（5.377 ± 0.057）,the LRRFIP1 protein level in high concentration group of cells were significantly higher than the control group（P〈0.05）,high concentration group of cells with ALDH1L1 gene was trended from partial methylation to completely methylation,the expressions of ALDH1L1 mRNA level in each groups were（1.023 ± 0.254）,（5.046 ± 0.603）,（2.259 ± 0.030）,（7.616 ± 1.910）,the ALDH1L1 mRNA level in each group was significantly higher than the control group（P〈0.05）,the expressions of ALDH1L1 protein level in each groups were（0.725 ± 0.014）,（0.913 ± 0.033）,（1.142 ± 0.010）,（1.334 ± 0.047）,the ALDH1L1 protein level in each concentration groups were significantly increased（P〈0.05）.Conclusion At some concentration point,MNNG significantly affects the methylation of ALDH1L1 and the expressions of p16 and FHIT on Kazakh normal esophageal epithelial cells.