Molecular characterization of the human PSMD4 gene

The ubiquitin-proteasome system (UPS) plays a critical role in the healthy progression of cellular processes by regulating protein homeostasis. The PSMD4 gene encodes one of the subunits of the 26S proteasome in UPS, which serves as the major ubiquitin receptor. In addition, the PSMD4 gene encodes the angiocidin protein with an additional three amino acids at the carboxyl terminus. PSMD4 and angiocidin proteins share similar sequences and have common binding interactions. There is no study in the literature revealing the role of the PSMD4 gene in pancreatic cancer. In this study, the angiocidin gene was obtained from PSMD4 gene by directed mutagenesis and both genes were cloned into pEGFP-C1 vector. PSMD4 and angiocidin genes were transferred into PANC-1 and HUVEC cells by transient transfection. Overexpression of genes was confirmed by real-time PCR, western blot, flow cytometry, and GFP analyses. Overexpression of PSMD4 and angiocidin increases proliferation, migration and colony formation ability in PANC-1 and HUVEC cells. In addition, the effect of overexpression on the expression of some genes associated with EMT and apoptosis processes was determined at the mRNA level. Also, partial silencing of PSMD4 gene in PANC-1 and HUVEC cells was performed using shRNA. PSMD4 gene silencing was confirmed by real-time PCR and immunofluorescence analyses. PSMD4 silencing reduces cell proliferation, migration, and colony formation ability in PANC-1 cells while increasing it in HUVEC cells. In the continuation of the study, it was determined that VEGF cytokine increased PSMD4 gene expression at mRNA and protein level in PANC-1 and HUVEC cells. VEGF was shown to affect PSMD4 gene regulation via P38 MAP kinase, JNK, MEK and PI3K pathways at the mRNA and protein level. Finally, the transcriptional activity of three promoter fragments of the PSMD4 gene in PANC-1 and HUVEC cells was determined. We foresee that the information obtained as a result of this thesis study will contribute to the development of treatments targeting the PSMD4 gene.