苯并芘促进FHIT、CDH13基因启动子区甲基化及机制研究  

Benzo(a)pyrene promotes the methylation of the promoter regions of FHIT and CDH13 genes and related mechanism

作　　者：武岳 张梦迪 李君 王冬雪[2,6] 白图雅 吕晓丽 胡玉霞 高峰 周树宏[4,5] 李京慧 范兴业 常福厚 王光 WU Yue;ZHANG Meng-di;LI Jun;WANG Dong-xue;BAI Tu-ya;LV Xiao-li;HU Yu-xia;GAO Feng;ZHOU Shu-hong;LI Jing-hui;FAN Xing-ye;CHANG Fu-hou;WANG Guang(The Affiliated Hospital of Inner Mongolia Medical University,Hohhot 010020;School of Pharmacy,Inner Mongolia Medical University,Hohhot 010110;Research Center for New Drug Safety Evaluation,Hohhot 010110;Research Center for New Drug Screening Engineering,Inner Mongolia Autonomous Region,Hohhot 010110;School of Basic Medicine,Inner Mongolia Medical University,Hohhot 010110;Institute of Interdisciplinary Sciences,Shanghai University of Traditional Chinese Medicine,Shanghai 201210)

机构地区：[1]内蒙古医科大学附属医院,呼和浩特010020 [2]内蒙古医科大学药学院,呼和浩特010110 [3]内蒙古医科大学新药安全评价研究中心,呼和浩特010110 [4]内蒙古自治区新药筛选工程研究中心,呼和浩特010110 [5]内蒙古医科大学基础医学院,呼和浩特010110 [6]上海中医药大学交叉科学研究院,上海201210

出　　处：《中南药学》2022年第9期2041-2045,共5页Central South Pharmacy

基　　金：内蒙古自治区高等学校科学研究项目(No.NJZY21593);内蒙古医科大学“三位一体”创新创业培育项目(No.SWYT2021034);内蒙古医科大学大学生科技创新“英才培育”项目(No.YCPY2022098)。

摘　　要：目的 探讨苯并芘(BaP)对脆性组氨酸三联体(FHIT)、T-钙黏蛋白(CDH13)基因启动子区甲基化的影响及可能机制。方法 选取DNA甲基转移酶抑制剂5-氮杂-2’-脱氧胞苷作为阳性对照组,采用甲基化特异性PCR(MSP)法检测不同浓度BaP(0、0.1、1、10 μmol·L^(-1))作用于HepG2细胞后对FHIT、CDH13基因甲基化的影响;采用RT-qPCR、Western blot法检测BaP作用于HepG2细胞后对FHIT、CDH13基因以及DNA甲基化转移酶1(DNMT1)、转录因子Sp1的mRNA与蛋白表达的影响。结果 与对照组相比,阳性对照组中CDH13、FHIT基因呈现非甲基化状态,随着BaP浓度的增加,FHIT、CDH13基因甲基化条带(M条带)亮度逐渐增强,非甲基化条带(U条带)亮度逐渐减弱,MSP法检测结果表明BaP可能有促进FHIT、CDH13基因甲基化的作用。RT-qPCR与Western blot法检测结果显示随着BaP浓度的增加,FHIT、CDH13基因的mRNA与蛋白表达量逐渐减少,与对照组相比,BaP中、高剂量组中FHIT、CDH13的表达显著降低(P<0.05);DNMT1、Sp1 的mRNA与蛋白表达显著增加(P<0.05)。结论 BaP具有促甲基化的作用,其机制可能是通过促进Sp1,进而促进DNMT1的表达,使DNMTs活性被增强或浓度过大,增强了原有的甲基化状态,从而促进FHIT、CDH13基因启动子区甲基化。Objective To determine the effect and mechanism of benzo(a)pyrene (BaP) on the methylation of promoter regions of fragile histidine triad (FHIT) and T-cadherin (CDH13) genes.Methods DNA methyltransferase inhibitor 5-Aza-2’-deoxycytidine was selected as the positive control group.Methylation specific polymerease chain reaction (MSP) was used to detect the effect of different concentrations of BaP (0,0.1,1,and 10 μmol·L^(-1)) on the methylation of FHIT and CDH13 genes on HepG2 cells.RT-qPCR and Western blot were used to detect the effect of different concentrations of BaP (0,0.1,1,and 10 μmol·L^(-1)) on HepG2 cells and further on the mRNA and protein expression of FHIT,CDH13,DNMT1,and Sp1.Results Compared with the control group,CDH13 and FHIT genes showed non-methylated status in the positive control group.With the increase of BaP concentration,the brightness of FHIT and CDH13 gene methylation bands gradually increased,while the brightness of unmethylated bands (U bands) gradually decreased.MSP showed that BaP promoted the methylation of FHIT and CDH13 genes.RT-qPCR and Western blot showed that with the increase of BaP concentration the mRNA and protein expressions of FHIT and CDH13 genes gradually decreased.Compared with the control group,the expressions of FHIT and CDH13 genes in the BaP medium and high dose groups were significantly decreased (P <0.05);the mRNA and protein expressions of DNMT1 and Sp1 greatly increased (P <0.05).Conclusion BaP can promote the methylation of FHIT and CDH13 genes promoter regions,whose mechanism is to promote the expression of DNMT1 by promoting Sp1.When the activity of DNMTs is enhanced or the concentration is too high,the original methylation is enhanced.

关 键 词：苯并芘 DNA甲基化 脆性组氨酸三联体 T-钙黏蛋白 

分 类 号：R96[医药卫生—药理学]