Exploring The Role Of PNPO On Cellular Proliferation In Multiple Myeloma

Background and ObjectiveMultiple Myeloma(MM)is a malignant plasmacyte proliferative disease,the second most common hematologic malignancy,mainly occurring in the elderly.Its clinical symptoms mainly include hypercalcemia,renal failure,anemia and bone disease[1].Although bortezomib,capfezomib and other chemotherapeutic drugs have been widely used in clinical practice,MM recurrence can also occur[2].Therefore,it is one of the urgent problems to find out the molecular mechanism that effectively regulates the development of MM.Pyridoxine-5’-phosphate oxidase(PNPO)is a gene that is associated with poor prognosis in patients with multiple myeloma screened from the clinical database.This study aims to elucidate the proliferation-promoting effect of PNPO on MM cells and the related specific molecular mechanism through in vivo and in vitro experiments,so as to provide a new molecular target for the clinical diagnosis and treatment of MM.Methods1.The PNPO gene with high expression in MM was screened by MM database,and the relationship between PNPO gene and the poor prognosis and survival rate of MM patients was analyzed.2.Western blot(WB)was used to verify the protein expression level of PNPO in MM cell lines H929,ARP1,XGI,OCI and CAG.The lentivirus was packaged using PNPO-cDNA or shRNA plasmid.Arp1 and CAG cells were transfected with lentivirus and puromycin was used to select stable PNPO-overexpressed or PNPOKD cell lines.WB confirmed the successful construction of PNPOOE or PNPOKD cell lines.3.The effect of PNPOOE on the proliferation of MM cells was detected by MTT assay.The effect of PNPOOE on the cell cycle of MM was detected by cytometry.The expression of Cyclin B1 protein in PNPOOE cells was detected by WB.4.The effect of PNPOKD on the proliferation of MM cells was detected by MTT assay.The effect of PNPOKD on the apoptosis of MM cells was detected by cytometry.WB detected the expression of PARP,Cyclin B1,Caspase3 and Cleaved Caspase3 in PNPOKD cells.5.Transcriptome sequencing was performed on both PNPOOE cells and Wild type(WT)cells,and differentially expressed genes were analyzed.The protein expressions of β-catenin,IL6 and CDK14 in PNPOOE or PNPOKD cells were detected by WB.The expression of β-catenin protein was detected by stimulating ARP1 and CAG MM cells of WT/PNPOKD with Wnt3a cytokines.6.The results of protein spectrum showed that the PNPO proteins were DVL3 and Cyclin B1.The results of mass spectrometry also indicated that methionine in DVL3 protein was oxidized.Immunoprecipitation experiments showed that there was an interaction between PNPO and DVL3 protein in PNPOOE cells,and the binding between PNPO and DVL3 protein decreased after the addition of GSH in PNPOOE cells.After co-transfection of Flag-PNPO plasmid and HA-DVL3 plasmid in 293T cells,there was an interaction between PNPO and DVL3,and the addition of GSH could effectively reduce the interaction between PNPO and DVL3.Results1.Database shows that PNPO gene expression in MM patients is significantly higher than that in patients with Monoclonal gammopathy of undetermined significance(MGUS)and Normal plasma(NP)(P<0.05);In the Assessment of Proteasome Inhibition for Extending Remission(Apex)clinical trial results,the survival rate of patients with high PNPO expression is significantly lower than that of patients with low PNPO expression.Similarly,Kaplan Meier survival analysis shows that high expression of PNPO is associated with lower survival in patients treated with Total Therapy2(TT2).2.WB experiment proved that the stable cell lines of PNPOOE were successfully constructed.MTT assay showed that PNPOOE promoted the proliferation of MM cells.Flow cytometry showed that PNPOOE significantly increased the ratio of G2/M phase,indicating increased cell proliferation.WB assay results showed that the expression of Cyclin B1 was up-regulated in PNPOOE cells.3.WB experiment proved that the stable cell lines of PNPOKD were constructed successfully.MTT assay showed that PNPOKD significantly inhibited the proliferation of MM cells.Flow cytometry showed that PNPOKD could promote the apoptosis of MM cells.WB further demonstrated that in PNPOKD cells,PARP and Cleaved Caspase3 expression were up-regulated,while Cyclin B1 expression was down-regulated.4.Transcriptome sequencing data of PNPOOE cells revealed three differentially expressed genes:TCF7L2,IL6 and CDK14.WB assay showed that PNPOOE or PNPOKD could increase or inhibit the protein expression levels of β-catenin,IL6 and CDK14.In addition,Wnt3a cytokine stimulation can up-regulate the expression of β-catenin protein in PNPOOE cells.5.The results of protein spectrum showed that the PNPO proteins were DVL3 and Cyclin B1.The results of mass spectrometry also indicated that methionine in DVL3 protein was oxidized.Immunoprecipitation experiments showed that there was an interaction between PNPO and DVL3 protein in PNPOOE cells,and the binding between PNPO and DVL3 protein decreased after the addition of GSH in PNPOIE cells.After co-transfection of Flag-PNPO plasmid and HA-DVL3 plasmid in 293T cells,there was an interaction between PNPO and DVL3,and the addition of GSH could effectively reduce the interaction between PNPO and DVL3.ConclusionsThe high expression of PNPO gene is associated with poor prognosis in MM patients.The overexpression of PNPO can promote the proliferation of MM cells and the development of MM cell cycle.PNPO promoted the proliferation of MM cells by oxidizing DVL3 protein.PNPO can be used as a new molecular target for clinical diagnosis and treatment of MM.