The luciferase reporter system of the MMP12 endogenous promoter for investigating transcriptional regulation of the human MMP12 gene
The luciferase reporter system of the MMP12 endogenous promoter for investigating transcriptional regulation of the human MMP12 gene
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MMP12内源启动子的荧光素酶报告系统用于研究人类MMP12基因的转录调控
DOI：
10.1016/j.ejbt.2019.12.003
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发表时间：
2020
影响因子：
2.7
通讯作者：
Xia Haibin
中科院分区：
文献类型：
--
作者：
Du Chunhua;Wu Yanqiu;Ju Yurong;Zhao Junli;Yang Peiyan;Mao Qinwen;Xia Haibin
背景基质金属蛋白酶12（MMP 12）是MMPs的一个成员，在细胞外基质成分降解、病毒感染、炎症、组织重塑和肿瘤发生等方面发挥重要作用。为探讨MMP 12基因的转录调控，利用CRISPR/Cas9技术构建了一株对MMP 12启动子敏感的荧光素酶报告基因HEK 293细胞系。测序结果表明，该细胞株基因组的一个等位基因被证实具有荧光素酶基因的定点插入，另一个等位基因被证实具有额外的48 bp插入。通过将转录因子STAT 3、AP-1和SP-1应用于该细胞系，进一步证明该细胞系是内源性MMP 12启动子的敏感报告基因。然后用生物活性小分子文库筛选报告细胞系，通过荧光素酶活性测定发现小分子丹参酮I在HEK 293-MMP 12-T2 A-luciferase-KI细胞系中显著抑制MMP 12基因的转录活性，进一步证实了该荧光素酶基因敲除物可抑制野生型HEK 293细胞中MMP 12 mRNA的表达。报告系统的建立将有助于研究MMP 12基因的转录调控和筛选针对MMP 12基因的药物。如何引用：杜C，吴Y，鞠Y，等. The luciferase reporter system of the MMP 12 endogenous promoter for investigating transformal regulation of human MMP 12 gene. 43.第43章. https://doi.org/10.1016/j.ejbt.2019.12.003.
BackgroundMatrix metalloproteinase 12 (MMP12), a member of MMPs, can take lots of roles including extracellular matrix component degradation, viral infection, inflammation, tissue remodeling and tumorigenesis. To explore the transcriptional regulation of MMP12 gene, a sensitive luciferase reporter HEK293 cell line for endogenous MMP12 promoter was generated by CRISPR/Cas9 technology.ResultsThe HEK293-MMP12-T2A-luciferase-KI cell line was successfully established by CRISPR/Cas9 technology. The sequencing results indicated that one allele of the genome was proven to have a site-directed insertion of luciferase gene and another allele of the genome was confirmed to have additional 48 bp insertion in this cell line. The cell line was further demonstrated to be a sensitive reporter of the endogenous MMP12 promoter by applying transcription factors STAT3, AP-1 and SP-1 to the cell line. The reporter cell line was then screened with bioactive small molecule library, and a small molecule Tanshinone I was found to significantly inhibit the transcriptional activity of MMP12 gene in HEK293-MMP12-T2A-luciferase-KI cell line by luciferase activity assay, which was further confirmed to inhibit the expression of MMP12 mRNA in wild-type HEK293 cells.ConclusionsThis novel luciferase knock-in reporter system will be helpful for investigating the transcriptional regulation of MMP12 gene and screening the drugs targeting MMP12 gene.How to cite:Du C, Wu Y, Ju Y, et al. The luciferase reporter system of the MMP12 endogenous promoter for investigating transcriptional regulation of the human MMP12 gene. Electron J Biotechnol 2020;43. https://doi.org/10.1016/j.ejbt.2019.12.003.
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