The Role Of PPFIA1 And The Identification Of The Phosphorylation Signaling Molecules And Interactive Proteins In Chronic Myelogenous Leukemia(CML) Cells

Background:We have previously revealed that mi R-181 a have multiple targets in CML by combining high-throughout microarray and bioinformatics. The preliminary result indicated that PPFIA1, a direct target of mi R-181 a, might function as oncogene. However, the exact role of PPFIA1 in CML is still unclear.Objective:Our goal is to investigate the roles of PPFIA1 and their phosphorylation signaling molecules and interactive proteins in CML cells. This study is expected to provide novel insights into the role of PPFIA1 and the targeted therapeutic in CML.Methods:1. A stable subline of K562 cells overexpressing PPFIA1 protein is established by PPFIA1 lentiviral particles and western blot asay detected the relative expression level of PPFIA1 protein post- transfection with PPFIA1 lentiviral particles.2. Transfecting PPFIA1-si RNA into K562 cells by LipofectamineTM 3000.3. The sensitivities of PPFIA1 in K562 cells on TKIs were determined by CCK8 assay.4. The colony-forming activity of PPFIA1 in K562 cells was tested by semi-solid culture medium of Methylcellulose.5. The migration and invasion activity of PPFIA1 in K562 cells was tested by transwell assays.6. Phosphorylated proteins were detected by Protein microarray technology and western blot.7. Establishing a stable subline of K562 cells overexpressing flag-PPFIA1 fusion protein and 293 T cells overexpressing flag-BCR/ABL fusion protein.8. Mass spectra identified those proteins from immunoprecipitated products by flag-PPFIA1 and flag-BCR/ABL protein. The Dicovery Studio software dock interactve proteins with PPFIA1 and BCR/ABL9. Western blot confirmed the immunoprecipitation(IP) products.10. CCK8 assay and semi-solid culture medium of Methylcellulose determined the effect of PARP1 inhibitor, olaparib, in K562-Lenti V PPFIA1 cells.Results:1. The relative expression of PPFIA1 protein in K562 cell of transfection with PPFIA1 lentiviral particles was higher by Western Blot assay.2. The overexpression of PPFIA1 protein significantly decreased the sensitivities of TKIs and promoted the abilities of migration, invasion and colony-forming ability in K562 cells.3. The low-expression of PPFIA1 protein significantly promoted the sensitivities of TKIs and decreased the abilities of migration, invasion and colony-forming ability in K562 cells.4. The number of phosphorylated proteins level decreased to over 20% in K562 cells is 28, especially the c-KIT(Tyr721) reaching 76%. And PPFIA1 protein can activating several signaling pathways such as C-KIT, AKT and ERK1/2 in K562 and KCL22 cells were validated by western blot.5. Proteins NCOA5 and PARP1 have been found in the two IP products both flag-PPFIA1 and flag-BCR/ABL proteins by Mass spectra.6. PPFIA1 can dock with PARP1 and NCOA5 protein using Discovery Studio 4.5 Client software. The results by Docking and Co-IP showed that PPFIA1 can directly interact with PARP1. Olaparib, a PARP1 inhibitor, significantly reduce cell vibility and colony-forming ability in K562-Lenti V PPFIA1 cell7. The overexpression of PPFIA1 significantly upregulated the expression level of PARP1, P65 and C-KIT phosphorylation protein. The targeted nhibition of PPFIA1 effectively downregulated the expression level of PARP1, P65 and C-KIT phosphorylation protein.Conclusion:1. PPFIA1 might acted as an oncogene in CML, and the targeted inhibition of PPFIA1 could reduced malignant progression of CML cells2. PPFIA1 regulate downstream phosphorylation signaling molecules of 28, in particular C-KIT.3. PPFIA1 finally activates the C-KIT signaling pathway by interacting with PARP1 protein, then promoting p65 transcription, and expands malignant progression of CML cells.