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

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

陈艳1， 邓彦超2,3， 黄思语1

(1新疆医科大学公共卫生学院，2新疆医科大学第一附属医院胸外科，3新疆食管癌研究所， 乌鲁木齐830011)

摘要: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、3 μg/mL MNNG的培养基中24 h，分别为对照组、0.75 μg/mL组、1.50 μg/mL组、3.00 μg/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,0.75 μg/mL组和3.00 μg/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，1.50 μg/mL和3.00 μg/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。

关键词：哈萨克族； 食管上皮细胞； LRRFIP1； ALDH1L1

中图分类号：R735.1文献标识码：A

doi:10.3969/j.issn.1009-5551.2015.05.008

[收稿日期：2014-09-22]

Effects of MNNG on methylation and expression of LRRFIP1, ALDH1L1

on Kazakh normal esophageal epithelial cells

CHEN Yan1, DENG Yanchao2,3, HUANG Siyu1

(1PublicHealthCollege,XinjiangMedicalUniversity,2DepartmentofChestSurgery,

TheFirstAffiliatedHospitalofXinjiangMedicalUniversity,3XinjiangInstituteforEsophageal

CancerResearch,Urumqi830011，China)

Abstract:ObjectiveTo investigate the effects of MNNG on methylation and expressions of LRRFIP1 and ALDH1L1 on Kazakh normal esophageal epithelial cells. MethodsMethylation 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. ResultsCompared 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). ConclusionAt some concentration point, MNNG significantly affects the methylation of ALDH1L1 and the expressions of p16 and FHIT on Kazakh normal esophageal epithelial cells.

Key words: Kazakh; esophageal epithelial cells;LRRFIP1;ALDH1L1

亚硝胺类化合物属亚硝基化合物，包括亚硝胺与亚硝酞胺两类。目前为止已发现的亚硝胺有300多种，其中90%以上的亚硝胺化合物对动物有致畸、致突变及致癌作用[1]。N-亚硝胺类烷化剂能诱导多种疾病的发生。研究结果表明，N-亚硝胺类化合物是诱发人类癌症的原因之一[2-3]。N-甲基-N′-硝基-N-亚硝基胍(MNNG)是一种人工合成的亚硝胺类化合物，常被用于研究亚硝胺类化合物诱发肿瘤的工具。动物实验证明，MNNG可以导致多种肿瘤的发生[4-5]。富含亮氨酸重复序列相互作用蛋白1[(leucinerich repeat(in FLII) interacting protein 1，LRRFIP1)]基因位于人类17号染色体上，是肿瘤坏死因子受体(TNFR)超家族的信号组成成分[6]。醛脱氢酶1家族L1基因(aldehyde dehydrogenase 1 family member L1，ALDH1L1)位于人类3号染色体的长臂上(3q21.2)，参与细胞增殖的调节，是存在于细胞质中的一个与叶酸代谢有关的酶基因[7]。……