CYP3A4内含子10对CYP3A4基因表达的增强子作用

王以婷; 杨卫红; 赵云龙; 曾昭书; 张莉蓉

CYP3A4内含子10对CYP3A4基因表达的增强子作用

1. 郑州大学基础医学院法医学系
2. 焦作市第二人民医院病理科
3. 郑州大学基础医学院药理学系

基金项目:

基金：国家自然科学基金资助项目 (81173127); 河南省杰出青年基金资助项目 (074100510020); 河南省教育厅基础研究基金资助项目 (13A310663); 河南省科技厅基础与前沿技术研究计划基金资助项目 (142300410206); 河南省科技攻关计划基金资助项目 (162102310523);

计量
- 文章访问数: 1640
- HTML全文浏览量: 593
- PDF下载量: 157
- 被引次数: 0
出版历程
- 收稿日期: 2016-09-20

The Enhancer Role of CYP3A4 Intron 10 in CYP3A4 Gene Expression

Funds:

摘要

摘要: 目的探讨CYP3A4内含子10及其内SNP CYP3A4*1G对CYP3A4基因表达的调控作用.方法构建CYP3A4启动子启动转录的CYP3A4真核表达质粒, 将CYP3A4内含子10 (CYP3A4*1G野生型/突变型) 正向或反向插入该质粒的启动子上游, 从而构建出不同基因型、不同方向的CYP3A4内含子10调控的CYP3A4真核表达质粒.将构建好的各质粒分别转染入Hep G2细胞, 应用实时荧光定量PCR方法检测CYP3A4 m RNA表达水平.结果构建的重组质粒经酶切验证与测序鉴定, 插入片段的序列和方向与预期完全一致.无论CYP3A4*1G野生型还是突变型, 反向组CYP3A4 m RNA表达水平均高于正向组 (P<0.01) ;而CYP3A4内含子10无论是正向还是反向, CYP3A4*1G野生型组CYP3A4 m RNA表达水平均高于突变型组 (P<0.01) .此外, 正向突变组CYP3A4 m RNA表达水平低于阳性对照组, 不但没有增强CYP3A4启动子的转录作用反而有所减弱.结论CYP3A4内含子10有类似于增强子的功能, 能够影响CYP3A4启动子对CYP3A4基因的转录, 且具有方向性和CYP3A4*1G等位基因依赖性.
- 内含子 /
- CYP3A4*1G /
- 真核表达载体
Abstract: Objective To explore the regulation of CYP3A4 gene expression by CYP3A4 intron 10 and CYP3A4*1G.Methods CYP3A4 eukaryotic expression vector which promoted by CYP3A4 promoter was constructed.The forward and reverse full-length segments of CYP3A4 intron 10 (with CYP3A4*1G allele wild/mutant type) were ligated into the recombinant eukaryotic expression vector above at upstream of CYP3A4 promoter to construct CYP3A4 eukaryotic expression vector which promoted by CYP3A4 promoter and regulated by CYP3A4 intron 10 with different genotypes and directions.The vectors were transfected into Hep G2 respectively.The levels of CYP3A4 m RNA were detected by quantitative real-time PC.Results These insertion sequences and their directions were exactly correct.The expression level of CYP3A4 m RNA was higher when CYP3A4 intron 10 was inthe forward than in the reverse direction (P<0.01) ;the wild group showed higher CYP3A4 m RNA level than the mutant group (P<0.01) ;while the forward mutant group showed lower CYP3A4 m RNA level than the positive control group.Conclusion CYP3A4 intron 10 is like an enhancer functionally, which could influence the transcription of CYP3A4 promoter depending on its direction and regulation by CYP3A4*1G allele.
- Intron /
- CYP3A4*1G /
- Eukaryotic expression vector

HTML全文

参考文献(21)

[1]	[1]WANG D, GUO Y, WRIGHTON S A, et al.Intronic polymorphism in CYP3A4 affects hepatic expression and response to statin drugs[J].Pharmacogenomics, 2011, 11 (4) :274-286.
[2]	[2]DROGEMOLLER B, PLUMMER M, KORKIE L, et al.Characterization of the genetic variation present in CYP3A4in three South African populations[J].Frontiers in genetics, 2013, 4 (17) :1-17.
[3]	[3]GAO Y, ZHANG L R, FU Q.CYP3A4*1G polymorphism is associated with lipid-lowering efficacy of atorvastatin but not of simvastatin[J].European Journal of Clinical Pharmacology, 2008, 64 (9) :877-882.
[4]	[4]ZHANG W, CHANG Y Z, KAN Q C, et al.CYP3A4*1G genetic polymorphism influences CYP3A activity and response to fentanyl in Chinese gynecologic patients[J].European Journal Of Clinical Pharmacology, 2010, 66 (1) :61-66.
[5]	[5]HE B X, SHI L, QIU J, et al.The effect of CYP3A4*1G allele on the pharmacokinetics of atorvastatin in Chinese Han patients with coronary heart disease[J].Journal of Clinical Pharmacology, 2014, 54 (4) :462-467.
[6]	[6]YUAN J J, HOU J K, ZHANG W, et al.CYP3A4*1G Genetic Polymorphism Influences Metabolism of Fentanyl in Human Liver Microsomes in Chinese Patients[J].Pharmacology, 2015, 96 (1-2) :55-60.
[7]	[7]MIURA M, SATOH S, KAGAYA H, et al.Impact of the CYP3A4*1G polymorphism and its combination with CYP3A5 genotypes on tacrolimus pharmacokinetics in renal transplant patients[J].Pharmacogenomics, 2011, 12 (7) :977-984.
[8]石伟龙, 唐惠林, 翟所迪.CYP3A4*1G基因多态性对肾移植受者他克莫司给药剂量及血药浓度影响的系统评价[J].中国临床药理学杂志, 2015, 31 (04) :292-296.
[9]	[9]YANG W H, ZHAO D Z, HAN S N, et al.CYP3A4*1G regulates CYP3A4 intron 10 enhancer and promoter activity in an allelic-dependent manner[J].International Journal of Clinical Pharmacology and Therapeutics, 2015, 53 (8) :647-657.
[10]赵云龙, 杨卫红, 闫良, 等.CYP3A4*1G依赖的CYP3A4内含子10启动的真核表达载体的构建[J].郑州大学学报 (医学版) , 2016, 51 (2) :153-157.
[11]	[11]CHATTERJEE S, MIN L, KARUTURI R K, et al.The role of post-transcriptional RNA processing and plasmid vector sequences on transient transgene expression in zebrafish[J].Transgenic Research, 2010, 19 (2) :299-304.
[12]	[12]ROSE A B.Intron-Mediated Regulation of Gene Expression[M].Current Topics in Microbiology and Immunology, 2008, 326 (6) :277-290.
[13]	[13]PAN Y Y, CHEN R, ZHU L, et al.Utilizing modified ubi1introns to enhance exogenous gene expression in maize (Zea mays L.) and rice (Oryza sativa L.) [J].Journal of Integrative Agriculture, 2016, 15 (8) :1716-1726.
[14]	[14]DE LA TORRE C M, FINER J J.The intron and 5’distal region of the soybean Gmubi promoter contribute to very high levels of gene expression in transiently and stably transformed tissues[J].Plant Cell Reports, 2015, 34 (1) :111-120.
[15]	[15]CHOKAS A L, BICKFORD J S, BARILOVITS S J, et al.A TEAD1/p65 complex regulates the eutherian-conserved Mn SOD intronic enhancer, e RNA transcription and the innate immune response[J].Biochimica et Biophysica Acta, 2014, 1839 (11) :1205-1216.
[16]	[16]POUND L D, HANG Y, SARKAR S A, et al.The pancreatic isletβ-cell-enriched transcription factor Pdx-1 regulates Slc30a8gene transcription through an intronic enhancer[J].Biochemical Journal, 2011, 433 (1) :95-105.
[17]	[17]KRIZEK B A.Intronic sequences are required for AINTEGUMENTA-LIKE6 expression in Arabidopsis flowers[J].BMC Res Notes, 2015, 8 (556) :1-9.
[18]	[18]SRIBUDIANI Y, METZGER M, OSINGA J, et al.Variants in RET associated with Hirschsprung's disease affect binding of transcription factors and gene expression[J].Gastroenterology, 2011, 140 (2) :572-582.
[19]	[19]OGURA Y, KOU I, MIURA S, et al.A Functional SNP in BNC2 Is Associated with Adolescent Idiopathic Scoliosis[J].Am J Hum Genet, 2015, 97 (2) :337-342.
[20]	[20]CAVALLI M, PAN GANG, NORD H, et al.Looking beyond GWAS:allele-specific transcription factor binding drives the association of GALNT2 to HDL-C plasma levels[J].Lipids Health Dis, 2016, 15 (18) :1-5.
[21]	[21]VISSER M, PALSTRA R J, KAYSER M.Allele-specific transcriptional regulation of IRF4 in melanocytes is mediated by chromatin looping of the intronic rs12203592 enhancer to the IRF4 promoter[J].Hum Mol Genet, 2015, 24 (9) :2649-2661.

相关文章(20)

[1]	张梁, 王保全, 雷喜锋, 王旭, 柯阳, 张玮. miR-29c-3p/IGF1分子轴对肝星状细胞活化，增殖和凋亡的作用机制, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20230926
[2]	朱中山, 杨洲, 江承川, 李小兵, 任斗, 黄橙, 张维薇, 李湘军, 赵顺利. 肺腺癌患者PLA2G1B表达情况与预后的相关性, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20220912
[3]	杨少华, 廖周俊, 胡晟, 洪茂林, 宋志美, 危群, 张小文. 真核翻译起始因子-5A2在肝内胆管癌中的表达及意义, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20210505
[4]	刘冲, 曹慧, 杨彩彩, 王震. 柚皮苷调控miR-199a-5p/ECE1分子轴促进骨损伤修复, 昆明医科大学学报.
[5]	金锦花, 李科岩, 张玲, 王玉慧, 邓晓明. 成纤维细胞生长因子受体FGFR1真核表达质粒的构建及其在HEK293T细胞的表达, 昆明医科大学学报.
[6]	朱柯颖, 蒋涛, 盘婕, 范志祥, 张璐, 张延洁, 吴艳瑞, 龙莉. CYP4A11基因rs9333025 A/G多态性在云南彝族人群中的分布, 昆明医科大学学报.
[7]	段君君, 方峰, 张小玉, 王江, 贾霄, 何颖红. WDR5真核表达载体的构建及其在LNCaP细胞中的稳定表达, 昆明医科大学学报.
[8]	王琨. shRNA-Bmi1重组载体构建及其对胆囊癌裸鼠移植瘤hTERT表达的影响, 昆明医科大学学报.
[9]	戴书颖. IL-4基因启动子SNP-1098T>G和-590C>T多态性与云南汉族人群HCV慢性感染的相关性研究, 昆明医科大学学报.
[10]	赵瑜. 人血管内皮生长因子A（VEGFA）的原核载体构建、蛋白表达及鉴定, 昆明医科大学学报.
[11]	方峰. ZAK-β蛋白激酶基因真核表达载体的构建, 昆明医科大学学报.
[12]	喻丽. TNIP1真核表达载体的构建及表达, 昆明医科大学学报.
[13]	徐盈. 脓毒症大鼠肿瘤坏死因子-a、内皮素-1、核因子kB表达与心肌损伤及药物影响的研究, 昆明医科大学学报.
[14]	赵留芳. pSIREN-HIF-1α/shRNA 表达载体的构建与鉴定, 昆明医科大学学报.
[15]	谷万港. HIV-1 整合酶真核表达载体的构建及在Hela细胞中的表达和定位, 昆明医科大学学报.
[16]	陈茜. CYP1B1 432C/G基因多态性与子宫内膜异位症易感性的相关研究, 昆明医科大学学报.
[17]	何颖红. IRF4逆转录病毒表达载体的构建及鉴定, 昆明医科大学学报.
[18]	田腊梅. 2种重组真核蛋氨酸酶原核体系表达及活性比较, 昆明医科大学学报.
[19]	毕城伟. Kringle5基因的克隆、原核表达载体构建和蛋白表达, 昆明医科大学学报.
[20]	詹辉. 改良型TAT -VP3融合蛋白的基因合成、原核表达载体构建及表达的实验研究, 昆明医科大学学报.