The Regulatory Mechanisms Of The Expression Of Receptor Tyrosine Phosphotase PTPRO

Human PTPRO is a member of the receptor-type protein tyrosine phosphatases (RPTPs) family, which is involved in podocyte function, axon pathfinding and tumor suppression. The expression of PTPRO varies under different conditions. For example, in kidney, PTPRO is expressed on the apical surface of the podocyte early in development and is present on the apical surface of podocyte foot processes in the mature phenotype; in brain, maximal expression of PTPRO is coincident with mid to late gestation and axonogenesis; in tumors, DNA hypermethylation-mediated silencing of PTPRO was reported in primary human tumors and cancer cell lines. However, we know little so far about the regulation mechanisms for PTPRO expression.This thesis describes the detailed study on the regulatory mechanisms of PTPRO expression. First, we discovered that PTPRO was a target of E2F1. E2F1 could up-regulate PTPRO mRNA level and activated PTPRO promoter. We also found that there existed two functional E2F1 binding sties in PTPRO promoter. By using EMSA and ChIP, we showed that E2F1 bound to PTPRO promoter in vitro and in vivo. Second, we showed that microRNA cluster miR-17-92, another target of E2F1, participated in PTPRO regulation. Bioinformatic analysis unraveled the existence of miR17-92 binding site in the 3'UTR of PTPRO mRNA, and luciferase reporter assay demonstrated the importance of miR-17-92 binding site in PTPRO post-transcriptional regulation. In addition, miR-17-92 could down-regulate PTPRO mRNA and protein level. Finally, we found that E2F1 and miR-17-92 were simultaneously involved in PTPRO regulation in cell cycle progression. We discovered that the promoter reporters inserted with PTPRO 3'UTR responded more weakly to E2F1 activation, implying that E2F1 first up-regulated PTPRO mRNA and then down-regulated PTPRO protein expression through up-regulating miR-17-92 transcription. Moreover, E2F1 overexpession indeed upregulated miR-17-5p, a member of miR-17-92 cluster. We then monitored PTPRO mRNA expression in synchronized HeLa cells, and found that PTPRO transcription was up-regulated at the S phase. Next, we examined the expression profiling of E2F1 and two miR-17-92 members, i.e., miR-17-5p and miR-20a, in synchronized HeLa cells. We found that both E2F1 mRNA and protein were up-regulated in G1 phase (2hr after released from double-thymidine arrest) whereas the expression of miR-17-5p was high in late S phase (6hr after released from double-thymidine arrest). These results demonstrated a positive correlation between E2F1 and PTPRO, and a negative correlation between miR-17-92 cluster, especially miR-17-5p and PTPRO. In addition, these results were coincided with our in vitro data that the PTPRO promoter activity was high in early S phase while the PTPRO 3'UTR reporter activity was low in late S phase in synchronized HeLa cells. Altogether, this study provides evidence that PTPRO gene is co-regulated by both E2F1 and miR-17-92.