Department of Respiratory Medicine, The First Affiliated Hospital of Zhengzhou University, No.1, Jianshe Road, Zhengzhou, 450052, China.
Mol Cell Biochem. 2013 Dec;384(1-2):1-6. doi: 10.1007/s11010-013-1775-7. Epub 2013 Aug 13.
Tissue inhibitor of metalloproteinases 1 (TIMP-1) is a glycosylated protein with multiple activities in the regulation of biological processes, such as cell growth and apoptosis as well as tumor invasion and metastasis. Bioinformatics analysis using TargetScan and miRanda suggested tissue inhibitors of TIMP-1 are among the targets of miR-1293. To confirm this, we cloned both wild-type and mutant TIMP-1 3'UTR fragments by overlap extension PCR, constructed the recombinant plasmids pGL3-TIMP-1-wt, -mut, and pcDNA 3.1(+)/TIMP-1-CDS and, respectively, co-transfected them into 293T cells with the miR-1293 inhibitor, mimics or the miR inhibitor-NC using a BTX ECM 2001 square-wave electroporator. We used a luciferase assay to investigate binding of miR-1293 to the 3'UTR of TIMP-1. Effects on the levels of the TIMP-1 protein were analyzed by Western blot experiments. The luciferase reporter assay showed a statistically significant (P < 0.05) upregulation of activity. Western blot analysis showed a significant increase of expression of the TIMP-1 gene co-transfected with the miR-1293 inhibitor, and demonstrated direct binding of miR-1293 to the 3'UTR of TIMP-1. In this study, we identified TIMP-1 as a novel direct target for miR-1293, which provides the basis for further study of the multifunctional mechanisms of miR-1293 and TIMP-1 in the regulation of a variety of diseases.
组织金属蛋白酶抑制剂 1（TIMP-1）是一种糖基化蛋白，在调节细胞生长、凋亡以及肿瘤侵袭和转移等生物学过程中具有多种活性。TargetScan 和 miRanda 的生物信息学分析表明，TIMP-1 的组织抑制剂是 miR-1293 的靶标之一。为了证实这一点，我们通过重叠延伸 PCR 克隆了野生型和突变型 TIMP-1 3'UTR 片段，构建了重组质粒 pGL3-TIMP-1-wt、-mut 和 pcDNA 3.1(+)/TIMP-1-CDS，并分别将它们与 miR-1293 抑制剂、模拟物或 miR 抑制剂-NC 一起使用 BTX ECM 2001 方波电穿孔仪转染 293T 细胞。我们使用荧光素酶 assay 来研究 miR-1293 与 TIMP-1 3'UTR 的结合。通过 Western blot 实验分析 TIMP-1 蛋白水平的影响。荧光素酶报告基因 assay 显示活性有统计学意义（P < 0.05）上调。Western blot 分析显示，与 miR-1293 抑制剂共转染的 TIMP-1 基因表达显著增加，并证明了 miR-1293 与 TIMP-1 3'UTR 的直接结合。在本研究中，我们确定 TIMP-1 是 miR-1293 的一个新的直接靶标，为进一步研究 miR-1293 和 TIMP-1 在调节多种疾病中的多功能机制提供了基础。
