Identification And Mechanism Of Bortezomib Resistance And Prognosis-related Gene NAMPT In Multiple Myeloma

Objective:Multiple myeloma(MM)is a common plasma cell tumor that results in the proliferation of malignant plasma cells and the overproduction of monoclonal immunoglobulin.Proteasome inhibitor(PI),represented by Bortezomib(BTZ),has always been the main treatment for MM,but most myeloma patients eventually develop drug resistance,leading to disease relapse.It is of great significance to explore the molecular mechanism of bortezomib resistance and find a possible cure for multiple myeloma patients with bortezomib resistance,so as to improve the treatment sensitivity and survival rate of MM patients.To find predictive biomarkers of BTZ resistance,further studies using raw MM samples and high-throughput sequencing data are necessary.Therefore,this study using bioinformatics methods,excavate potential molecular targets of resistance in MM,and use the GEO database data and clinical sample data to verify its feasibility,and then preliminarily target genes in the MM cell proliferation,apoptosis,and the role of bortezomib resistance mechanism,thus providing a new method for the clinical treatment of multiple myeloma.Methods:1.Microarray dataset GSE9782 was downloaded from GEO database,and DEGs of bortezomib sensitive and resistant MM patients were screened by GEO2 R online analysis tool.DAVID database was used to analyze the GO function,KEGG pathway and REACTOME pathway enrichment of DEGs.PPI network was constructed by using STRING database,and hub genes were further screened by Betweenness,Bottleneck and Stress in Cyto Hubba.Survival analysis of hub genes was conducted by Kaplan-Meier Plotter,and the target gene NAMPT was screened and identified by literature review.The gene expression data and clinical data of three microarray datasets GSE6477,GSE31162 and GSE24080 were downloaded from GEO database,and the expression of the selected target gene NAMPT in MM and its correlation with clinical characteristics and prognosis were initially verified.GSEA analysis was performed using another independent MM microarray dataset GSE19784,to explore the possible mechanism of NAMPT involved in MM cell function.2.Bone marrow samples and detailed clinical data were collected from 85 MM patients and 15 healthy hematopoietic stem cell transplantation donors hospitalized in the Department of Hematology,The Second Hospital of Shanxi Medical University from December 2018 to October 2020.q RT-PCR and Western blot were used to detect the relative expression levels of NAMPT m RNA and protein in bone marrow mononuclear cells of MM patients and healthy donors,and to explore the correlation between the expression of NAMPT and clinicopathological features and efficacy of MM patients.Kaplan-meier method was used to analyze the effect of NAMPT on PFS and OS in MM patients,and univariate and multivariate survival analyses were performed.3.The expression levels of NAMPT m RNA and protein in multiple myeloma cell lines MM1 R,MM1S,U266 and RPMI-8226 were detected by q RT-PCR and Western blot.The U266 cell line with the highest expression level of NAMPT was selected as the research object.Si RNA-NAMPT and negative control Si RNA was transfected into U266 cells.NAMPT was stably knocked out in U266 cells.p Max GFP vector was used to evaluate the transfection success rate of U266 cells.Western blot was used to detect the protein level of NAMPT in U266 cells with stable NAMPT knockout to verify the knockout effect.CCK8 method was used to detect the proliferation of U266 cells after transfection,and flow cytometry was used to detect the apoptosis rate of cells with and without NAMPT knockout.The recombinant plasmid Flag-NAMPT and empty vector Flag were transfected into U266 cells,respectively,and NAMPT was stably overexpressed in U266 cells.Western blot analysis was used to analyze the overexpression effect.CCK8 method was used to detect the proliferation of U266 cells after overexpression of NAMPT,and flow cytometry was used to detect the apoptosis rate of U266 cells in the presence and absence of stable overexpression of NAMPT.The effects of NAMPT on MM cell proliferation,apoptosis were confirmed from both forward and reverse aspects.4.JASPAR database was used to detect the upstream genome sequence of NAMPT,and a potentially conserved XBP1 binding region was found in the NAMPT promoter.The specific binding of XBP1 gene and NAMPT promoter gene fragments was confirmed by Ch IP analysis.XBP1 gene was knocked out and overexpressed in U266 cells,respectively.Western blot was used to analyze the effect of XBP1 gene knockout and overexpression,and q RT-PCR was used to detect the NAMPT m RNA levels before and after XBP1 gene knockout and overexpression,so as to explore the regulatory mechanism of the abnormal expression of NAMPT at the transcriptional level.5.XBP1-Si RNA,NAMPT-Si RNA and negative control Si RNA were transfected into U266 cells for 24 h,and then treated with bortezomib for 24 h.Cell proliferation of each group was determined by CCK8 method.In addition,Dimethyl Sulfoxide(DMSO)was used as negative control group.U266 cells were pretreated with IRE1α inhibitor MKC3946 for 4h and bortezomib for 24 h.Cell proliferation was determined by CCK8 method.The m RNA and protein expressions of apoptosis-related genes BCL2 and BAX were detected by RT-PCR and Western blot.Caspase-3/7 assay kit was used to analyze the activity of Caspase-3/7.Results:1.The online tool GEO2 R was used to screen out DEGs between patients with bortezomib sensitivity and drug resistance in the GSE9782 dataset,including 112up-regulated genes and 89 down-regulated genes.DEGs were analyzed for enrichment of GO function,KEGG and REACTOME pathway.GO function includes biological process(BP),molecular function(MF)and cellular component(CC).BP analysis suggested that DEGs was significantly enriched in translation initiation,m RNA degradation and metabolism,SRP-dependent cotranslation proteins targeting cell membranes.Cell components(CC)are mainly enriched in cytoplasmic ribosomes,ribosomal subunits and cytoplasmic parts.The main molecular functions(MF)involved ribosomal structural components,translation initiation factor activity,translation factor activity,RNA binding,cell adhesion,etc.KEGG pathway found that they were significantly enriched in ribosomal production,oxidative phosphorylation,RNA transport,NF-KB signaling,TNF signaling,and apoptosis.Enrichment analysis of REACTOME pathway suggested that up-regulated DEGs played an important role in MM resistance.We constructed a PPI network consisting of 201 nodes and 960 links,and further identified 7 Hub genes using Betweenness,Bottleneck and Stress methods,Including RPS27 A,RPS16,COX7 C,LCK,NAMPT,PTEN,and CXCL8.Hub genes survival analysis showed that only NAMPT was associated with overall survival in MM patients.Combined with PPI analysis,survival analysis and literature review,NAMPT was identified as the target gene of this study.Big data analysis of the microarray dataset GSE6477,GSE31162,and GSE24080 showed that the expression of NAMPT in newly diagnosed MM was significantly increased compared with normal plasma cells(P<0.001).The expression of NAMPT in relapsed MM cells was significantly higher than that in newly diagnosed MM cells(P<0.001).NAMPT expression was significantly correlated with β2-microglobulin(P=0.046),serum lactate dehydrogenase(P=0.012),and the number of focal lesions identified by magnetic resonance imaging(P=0.008).Event-free survival(P=0.037)and overall survival(P=0.009)were higher in patients with low NAMPT expression than in those with high NAMPT expression,and NAMPT expression was an independent prognostic factor for PFS and OS in MM patients(P=0.006,P=0.020).GSEA result suggested that NAMPT may affect proliferation of MM cells through m TORC1 signaling pathway.2.NAMPT m RNA expression in bone marrow of newly diagnosed and relapse MM patients was significantly higher than that of healthy donors(P<0.001).The expression of NAMPT m RNA in patients with relapse MM was significantly higher than that in patients with newly diagnosed MM(P<0.001),which was consistent with the expression of NAMPT protein.NAMPT expression was significantly up-regulated in patients with ISS stage III,elevated lactate dehydrogenase and C-reactive protein levels,P53 deletion,and a higher proportion of myeloma cells with adverse clinicopathological features(P<0.001).Compared with CR group,NAMPT m RNA expression was significantly up-regulated in PR,progression and relapse groups(P<0.001).The median PFS of patients with high NAMPT expression(14.9 months)was significantly shorter than that of those with low NAMPT expression(27 months,P<0.001).Median OS of patients with high NAMPT expression group(27.3 months)was significantly shorter than that of patients with low NAMPT expression group(39.1 months,P=0.048).Univariate and multivariate analyses showed that high NAMPT expression was associated with shorter PFS and OS.3.The m RNA and protein levels of NAMPT in MM cell lines MM1 R,MM1S,U266 and RPMI-8226 were significantly higher than those in normal bone marrow cells(P<0.001),and the expression level was the highest in U266 cells.Gene knockout effect detection showed that more than 80% of MM cells expressed green fluorescence signal24 h after transfection,indicating that transfection was successful.Compared with Si-NC group,the expression level of NAMPT protein in Si-NAMPT group was significantly decreased(0.89 ±0.06 vs 0.22±0.06,t=-24.42,P<0.001).Compared with Si-NC group,Si-NAMPT group significantly inhibited the proliferation of U266 cells after transfection for 24,48 and 72 hours(24h: 1.03±0.11 vs 0.62±0.08;48h: 1.62±0.11 vs 0.78±0.06;72h:2.37±0.18 vs 1.23± 0.14),the difference was statistically significant(P=0.006,P<0.001,P=0.001).Compared with Si-NC group,the apoptosis rate of U266 cells in Si-NAMPT group was significantly increased at 48 h after transfection [(35.43±1.67)% vs(53.42±0.25)%],and the difference was statistically significant(t=4.41,P<0.001).These results suggest that stable deletion of NAMPT can inhibit proliferation and induce apoptosis of U266 cells.Compared with Flag-NC group,the expression level of NAMPT protein in U266 cells in Flag-NAMPT group was significantly increased(0.12±0.08 vs1.18±0.07,t=16.42,P<0.001).With the extension of action time,cell proliferation in Flag-NAMPT group was significantly increased compared with Flag-NC group(24h:1.13±0.05 vs 1.52±0.09;48h: 1.60±0.24 vs 2.67±0.07;72h: 2.38±0.07 vs 4.93±0.24),the difference was statistically significant(P=0.003,P=0.002,P<0.001).Compared with the control group,the apoptosis rate of Flag-NAMPT group was significantly decreased[(32.77±1.39)% vs(21.92±1.56)%],the difference was statistically significant(t=-8.99,P=0.001).These results indicate that,contrary to the result of NAMPT gene knockout,the overexpression of NAMPT can promote the proliferation and inhibit the apoptosis of U266 cells.4.XBP1 regulates NAMPT expression at the transcriptional level.JASPAR database was used to analyze the upstream sequence of NAMPT genome.A potentially conserved XBP1 binding site was found in the promoter region of NAMPT from 1913 bp to 1926 bp.Chromatin immunoprecipitation(Ch IP)analysis showed that endogenous XBP1 could specifically bind to the upstream binding site of NAMPT gene in U266 cells.Compared with Flag-NC group,XBP1 protein expression level of U266 cells in Flag-XBP1 group was significantly increased,and Flag-XBP1 could significantly up-regulate NAMPT m RNA level(1.00±0.00 vs 6.13±0.41,t=21.368,P<0.001).The stable knockout of XBP1 resulted in a significant downregulation of NAMPT m RNA level in U266 cells(1.00±0.00 vs 0.32± 0.05,t=-20.52,P<0.001).These results suggest that the expression of NAMPT in U266 cells is regulated by XBP1 at the transcriptional level.5.Inhibition of IRE1α-XBP1-NAMPT axis increases the sensitivity of U266 cells to bortezomib.After treatment with bortezomib,the proliferation rate of U266 cells stably knocked out by XBP1 or NAMPT decreased to(62.73±3.62)% and(52.70±4.08)%,the differences were statistically significant compared with Si-NC group(P<0.001).Intracellular BCL2/BAX ratio plays an important role in determining the sensitivity of cell apoptosis.Activation of Caspase-3/7 can also be considered as an effect of U266cells’ sensitivity to bortezomib treatment.Compared with Si-NC group,the m RNA and protein levels of BAX in U266 cells with XBP1 and NAMPT knocked out were significantly increased,while the m RNA and protein levels of BCL2 were significantly down-regulated,Caspase-3 cleavage was significant and Caspase-3/7 activity was significantly increased(P<0.001).In addition,Dimethyl Sulfoxide(DMSO)as a negative control group,U266 cells were pretreated with MKC3946 for 4h and then treated with bortezomib for 24 h,the cell proliferation rate decreased significantly,which was(61.81±5.26)% of the control group,BAX m RNA and protein levels were significantly increased,BCL2 m RNA and protein levels were significantly down-regulated,Caspase-3cleavage was significant,Caspase-3/7 activity was sharply increased(P<0.001),consistent with the above results.These results suggest that inhibition of IRE1α-XBP1-NAMPT axis can induce U266 cells to be more sensitive to bortezomib and more susceptible to apoptosis.Conclusion:1.High expression of NAMPT may lead to bortezomib resistance in MM patients,which is associated with poor prognosis of MM patients and is expected to be used as a drug resistance and prognostic biomarker of MM.2.Compared with healthy controls,the expression level of NAMPT was significantly up-regulated in newly diagnosed and relapsed MM patients,which was associated with adverse clinical characteristics,efficacy and prognosis of MM patients,and was an independent prognostic risk factor for MM.3.High expression of NAMPT can promote MM cell proliferation and inhibit apoptosis.Mechanistically,NAMPT is regulated by IRE1α-XBP1 signaling pathway in U266 cells.Stable knockout of NAMPT expression or blocking of IRE1α-XBP1 pathway can significantly increase the sensitivity of U266 cells to bortezomib.