南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (3): 425-431.doi: 10.12122/j.issn.1673-4254.2022.03.16

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二甲双胍和LPS通过转录因子RUNX2调控NFATc2基因的转录

薛晓阳，李忠豪，赵 明   

  1. 南方医科大学第二临床医学院，广东 广州 510515；广东省医学休克微循环重点实验室，南方医科大学基础医学院病理生理学教研室，广东 广州 510515
  • 出版日期:2022-03-20 发布日期:2022-04-12

Metformin and lipopolysaccharide regulate transcription of NFATc2 gene via the transcription factor RUNX2

XUE Xiaoyang, LI Zhonghao, ZHAO Ming   

  1. Second School of Clinical Medicine, Southern Medical University, Guangzhou 510515, China; Key Lab of Medical Shock and Microcirculation Research of Guangdong Province, Department of Pathophysiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
  • Online:2022-03-20 Published:2022-04-12

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摘要： 目的 构建人T细胞活化核因子C2（NFATc2）基因启动子荧光素酶报告基因载体pGL3-NFATc2-promoter，检测其转录活性，探究二甲双胍和脂多糖对其转录活性的影响。方法 利用UCSC网站查找人NFATc2基因的启动子序列并设计上下游引物PCR扩增人NFATc2基因启动子片段；用限制性内切酶KpnⅠ和HindⅢ双酶切质粒pGL3-basic，将人NFATc2基因启动子片段插入到pGL3-basic质粒，重组质粒命名为pGL3-NFATc2-promoter。将pGL3-NFATc2-promoter与内参质粒pRL-TK共转染293F细胞，检测其荧光素酶活性。同时构建人NFATc2基因启动子不同片段长度的报告基因载体并进行荧光素酶活性检测，分别给予不同浓度的二甲双胍和脂多糖处理24 h后检测二甲双胍和脂多糖对NFATc2转录活性的影响。进一步突变NFATC2基因启动子上转录因子RUNX2的结合位点探究二甲双胍和脂多糖对NFATc2的转录调控作用。结果 研究成功构建了不同片段长度（2170、2077、1802、1651、1083、323 bp）的人NFATC2基因启动子荧光素酶报告基因载体pGL3-NFATc2-promoter，经酶切及测序鉴定完全正确。将不同片段长度的pGL3-NFATc2-promoter转染到293F细胞，发现pGL3-1651 bp具有最高转录活性，荧光素酶活性约为pGL3-2170 bp的3.3倍（1.8433±0.1457 vs 0.5467±0.0850）。中浓度（5 mmol/L）和高浓度（10 mmol/L）的二甲双胍分别上调pGL3-1651 bp的转录活性多达2.5、3倍（1.3467±0.1601 vs 0.8867±0.0321，1.8124±0.2771 vs 0.8867±0.0321）。不同剂量的脂多糖均能上调pGL3-1651 bp的转录活性不低于1.6倍（1.4813±0.0616 vs 0.8867±0.0321）。突变pGL3-1651 bp上的RUNX2结合位点后，二甲双胍和脂多糖上调的pGL3-1651 bp转录活性均受到抑制，转录活性下降（2.1667±0.1527 vs 1.233±0.1155；2.3667±0.2887 vs 1.1333±0.3786）。结论 pGL3-NFATc2-promoter在293F细胞中能被转录激活，并证实脂多糖和二甲双胍转录激活pGL3-NFATc2-promoter依赖于转录因RUNX2。

关键词: NFATc2；脂多糖；二甲双胍；RUNX2；报告基因

Abstract: Objective To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene. Methods The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn I and Hind III. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription. Results We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively. Conclusion pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3-NFATc2-promoter in a RUNX2-dependent manner.