Abstract 4558: PIN1 expression level is maintained by a negative feedback loop through regulation of Myc and miRNAs

Introduction: PIN1 is a peptidyl-prolyl-isomerase (PPIase) that binds specific motifs (serine/threonine preceding a proline) in proteins, thereby catalyzing cis/trans isomerization of the peptide bond between the phosphorylated serine/threonine and proline. As a result of such conformational changes, stability, sub-cellular localization, functional activity as well as protein-protein interactions of the PIN1-bound proteins may be altered. PIN1 has been demonstrated to play an important role in controlling various cellular processes such as cell proliferation, apoptosis and migration. Consequently, its expression has to be tightly controlled. Previously, we found that two miRNAs, miR-296-5p and miR-874-3p, served as negative regulators of PIN1. However, the mechanism regulating these two miRNAs remains to be defined. Methods: The online database-UCSC Genome Bioinformatics (NCBI/hg18) was used to search for the upstream regulator of miR-296-5p and miR-874-3p. Luciferase reporter assay was conducted to examine the effect of the identified regulator on the promoter regions of miR-296 and miR-874-3p. Quantitative polymerase chain reaction (Q-PCR) was used to detect the expression levels of miR-296-5p and miR-874-3p. Protein expression levels of the identified regulator and PIN1 were examined by western immunoblotting. Results: By screening the online database-UCSC, Myc was identified as a potential transcription factor for the miRNAs, miR-296-5p and miR-874-3p. As demonstrated by luciferase reporter assay, expression of Myc activated the promoters of miR-296-5p and miR-874-3p. Myc over-expression also increased the expression levels of these two miRNAs, which in turn led to down-regulation of PIN1. Interestingly, we found that over-expression of PIN1 increased the expression of Myc, miR-296-5p and miR-874-3p. We therefore hypothesized that PIN1 expression is maintained via a negative feedback loop of Myc-miRNAs-PIN1. Consistent with this hypothesis, we demonstrated that over-expression of exogenous EGFP-tagged PIN1 led to the enhancement of Myc, miR-296-5p and miR-874-3p expression, and thereby subsequently decrease the endogenous PIN1 level. Conclusion: Taken together, our results suggested that the expression level of PIN1 is maintained by a negative feedback loop through the regulation of Myc, miR-296-5p and miR-874-3p. Citation Format: Ka Wai Leong, Chi Wai Cheng, Yok Lam Kwong, Wai Choi Eric Tse. PIN1 expression level is maintained by a negative feedback l