转录因子GATA结合蛋白3对哮喘易感基因ADAM33表达的影响

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摘要目的:探讨转录因子GATA结合蛋白3(GATA binding protein 3,GATA3)对解整合素金属蛋白酶33(a disintegrin and metalloproteinase 33, ADAM33)基因启动子活性及mRNA表达的影响。方法: 以人正常肺上皮BEAS-2B细胞DNA为模板经PCR扩增得到ADAM33基因启动子区1 953 bp片段。通过双酶切、连接、转化等步骤将该片段克隆至pGL3-Basic载体构建ADAM33启动子重组质粒,并将其转染至人HEK293T、BEAS-2B细胞中；利用双荧光素酶报告基因技术检测HEK293T、BEAS2B细胞中所构建的质粒启动子活性。将HEK293T、BEAS-2B细胞分为pcDNA-GATA3组和pcDNA3.1对照组,分别转染GATA3过表达质粒和空白对照质粒,用双荧光素酶报告基因技术检测荧光素酶活性;用实时荧光定量PCR检测BEAS-2B细胞中ADAM33 mRNA相对表达水平。结果: 通过PCR、双酶切和测序鉴定,成功构建含ADAM33启动子片段的重组质粒;重组质粒的ADAM33启动子活性较pGL3-Basic对照组明显增高。与pcDNA3.1组相比,HEK293T、BEAS-2B细胞中pcDNAGATA3组ADAM33启动子区荧光素酶活性明显增高(P<0.05或P<0.01);BEAS-2B细胞中pcDNA-GATA3组ADAM33 mRNA相对表达水平明显增高(P<0.01)。结论: 转录因子GATA3上调哮喘易感基因ADAM33启动子活性及mRNA表达。

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	芮菲菲
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	郝忠分
	马世奇
	王雯
	束进

关键词 ：启动子活性, GATA结合蛋白3, 解整合素金属蛋白酶33, 哮喘, BEAS-2B细胞

Abstract：Objective: To investigate the effect of transcription factor GATA binding protein 3(GATA3) on promoter activity and mRNA expression of a disintegrin and metalloproteinase 33(ADAM33). Methods: The 1 953 bp fragment of ADAM33 promoter region was amplified by PCR based on human normal lung epithelial BEAS-2B cell DNA as template. The recombinant plasmid of ADAM33 promoter was cloned into pGL3-Basic vector by double digestion, ligation and transformation, and then transfected into HEK293T and BEAS-2B cells. The promoter activity of the constructed plasmid of ADAM33 gene in HEK293T and BEAS-2B cells were detected by double luciferase reporter gene technology. The HEK293T and BEAS-2B cells were divided into pcDNA-GATA3 group and pcDNA3-1 control group, respectively, and transfected with GATA3 overexpression plasmid and blank control plasmid,respectively; the luciferase activity was detected by dual luciferase reporter gene technology; the mRNA expression of ADAM33 in BEAS-2B cell was detected by real-time fluorescence quantitative PCR. Results: The recombinant plasmid containing ADAM33 promoter fragment was successfully constructed by PCR, double enzyme digestion and sequencing. Compared with the pcDNA31group, the luciferase activity in the ADAM33 promoter region of pcDNA-GATA3 group in HEK293T and BEAS2B cells was significantly increased(P<0.05 or P<0.01); the pcDNAGATA3 group showed higher level of the relative expression of ADAM33 mRNA(P<0.01). Conclusion: Transcription factor GATA3 up-regulates the promoter activity and mRNA expression of human asthma susceptibility gene ADAM33.

收稿日期: 2021-07-19

基金资助:镇江市社会发展项目(SH2020063)；镇江市卫生计生科技重点专项项目(SHW2017012)

通讯作者: 束进(通讯作者)，副主任医师，E-mail： zjshujin@163.com

作者简介: 芮菲菲(1996—)，女，硕士研究生；

引用本文:

芮菲菲, 杜娟, 郝忠分, 马世奇, 王雯, 束进. 转录因子GATA结合蛋白3对哮喘易感基因ADAM33表达的影响[J]. 江苏大学学报:医学版, 2022, 32(02): 131-135. RUI Feifei, DU Juan, HAO Zhongfen, MA Shiqi, WANG Wen, SHU Jin. Effect of transcription factor GATA binding protein 3 on the expression of asthma susceptibility gene ADAM33. Journal of Jiangsu University(Medicine Edition), 2022, 32(02): 131-135.

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http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2022/V32/I02/131

［1］WillisOwen S, Cookson W, Moffatt MF. The Genetics and Genomics of Asthma［J］. Annu Rev Genomics Hum Genet, 2018, 19: 223-246.
［2］Van Eerdewegh P, Little RD, Dupuis J, et al. Association of the ADAM33 gene with asthma and bronchial hyperresponsiveness［J］. Nature, 2002, 418(6896): 426-430.
［3］Tripathi P, Awasthi S, Gao P. ADAM metallopeptidase domain 33(ADAM33): a promising target for asthma［J］. Mediators Inflamm, 2014, 2014: 572025.
［4］Irvin C, Zafar I, Good J, et al. Increased frequency of dualpositive TH2/TH17 cells in bronchoalveolar lavage fluid characterizes a population of patients with severe asthma［J］. J Allergy Clin Immunol, 2014, 134(5): 1175-1186.e7.
［5］Shrine N, Portelli MA, John C, et al. Moderatetosevere asthma in individuals of European ancestry: a genomewide association study［J］. Lancet Respir Med, 2019, 7(1): 20-34.
［6］Keil T, Baldassi D, Merkel OM. Tcell targeted pulmonary siRNA delivery for the treatment of asthma［J］. Wiley Interdiscip Rev Nanomed Nanobiotechnol, 2020, 12(5): e1634.
［7］方丽萍,陈敏,张方,等. 解整合素—金属蛋白酶33在不同严重程度哮喘患者血清中的表达水平及其与气道慢性炎症的关系［J］. 医学研究生学报, 2017, 30(5): 521-524.
［8］Yang Y, Haitchi HM, Cakebread J, et al. Epigenetic mechanisms silence a disintegrin and metalloprotease 33 expression in bronchial epithelial cells［J］. J Allergy Clin Immunol, 2008, 121(6): 1393-1399, 1399.e1-14.
［9］Schwenger GT, Mordvinov VA, Sanderson CJ. Transcription factor GATA3 is involved in repression of promoter activity of the human interleukin4 gene［J］. Biochemistry (Mosc), 2005, 70(9): 1065-1069.
［10］Yamashita M, UkaiTadenuma M, Kimura M, et al. Identification of a conserved GATA3 response element upstream proximal from the interleukin13 gene locus［J］. J Biol Chem, 2002, 277(44): 42399-42408.
［11］鲁娜,白燕,赵晨,等. 槐杞黄对哮喘大鼠模型的作用及其机制研究［J］. 中国当代儿科杂志, 2020, 22(2): 171-176.