Identification of miR-1293 potential target gene: TIMP-1.

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.