The Role And Mechanism Of IL-32 And IL-32 Triggered Cytokines In Occurrence And Development Of Multiple Myeloma

Background:Multiple myeloma(MM)is a maligangt blood system disease which originates from the B cell lineage,characterized by accumulation of malignant plasma cells and increase of monoclonal globin.MM is closely associated with inflammation.Patients with auto-immune disease,history of infection,and other inflammatory diseases have higher incidence of MM.We previously demonstrated that MM cells and bone marrow stromal cells(BMSCs)could be modified by each other thus "co-evolution" to formulate a inflammatory microenvironment that benefit MM cells growth.In MM bone marrow microenvironment,BMSCs are the primary source of interleukin-6(IL-6)secretion,which promotes the proliferation and development of MM cells.Researches indicated that elevated expression of IL-6 in MM patients is positively assasiated with high tumor burden and the progression of disease.How the inflammation makes BMSCs secret IL-6,however,remained undocumented.Our subsequent study compared the differential secretion of peripheral blood between MM patients and normal people by cytokine array,and showed that interleukin 32(IL-32)is highly expressed in MM patients.IL-32,also named natural killer 4(NK-4),is a newly found inflammatory factor which have nine different isoforms.Accumalative evidience showed that IL-32 is a pro-inflammatory factor which triggers a massive amplification of inflammatory process resulting in the change of other inflammatory factor including IL-6,IL-10,and TNF-?.Studies in solid tumors,such as gastric cancers,pancreatic cancers and renal carcinomas,have found high expression of IL-32,suggesting that this cytokine is an independent prognostic factor.In this study,we explored the change of cytokine network under the action of IL-32,to find the effect of inflammatory cytokines in MM cell genetic instability and proliferation,apoptosis and the molecular mechanism.Objective1.To identify the expression of IL-32 in MM patients and clarify the primary source of IL-32 production in MM bone marrow microenvironment.2.To explore the role and function of IL-32 in triggering the changes of cytokine network in MM bone marrow microenvironment,to develop IL-32 knock downt MM cells to verify the effect of IL-32 in MM.3.To sduty some of the molecular mechanism involved in the changes of cytokines that induced by IL-32.4.To explore the effect of IL-32 on the MM cell biology behavior.Methods1.Compare the expression of IL-32 in MM patients and healthy donors by ELISA,study the expression of IL-32 in BMSCs,MM cell lines(RPMI8226,OPM-2,NCI-H929 and LP-1)and primary CD138-/CD138+ cells by immunofluorescence,RT-PCR.2.Stimulated BMSCs with recombinant IL-32?(rIL-32?)in both dose escalating manner and time escalating manner,detect the expression of IL-6,TNF-? and other cytokines by ELISA and cytokine array;develop IL-32 knock down MM cell lines by shRNA,build a BMSCs/MM co-culture system with or without IL-32 knock down in MM cells,ELISA was applied to detect the IL-6 expression in BMSCs.3.Develop PR3 knock down BMSCs by siRNA.Use rIL-32? stimulate BMSCs with or without PR3 knock down,in both dose escalating manner and time escalating manner,Western blot was applied to explore the changes in inflammation signaling pathway.4.Use CCK8,flow cytometry and Western blot to identify the MM cell biology behavior(proliferation,apoptosis and DNA damage)induced by the IL-32 mediated changes of cytokines.Subcutaneous injection of transfection MM cells to study IL-32 effect on myeloma cells in vivo by transplantation myeloma model in NOD/SCID mice.Results1.MM patients exhibited significantly higher secretion of soluble IL-32 compared to healthy controls in both bone marrow and peripheral blood.Immunofluorescence,qRT-PCR and ELISA showed that in MM bone marrow CD138+ cells had a relatively high expression of IL-32 compared to CD 13 8" cells.MM cell lines generally expressed IL-32.RPMI8226 and OPM2 have relatively high expression of IL-32,H929 cells and LP-1 have relatively low expression of it.2.rIL-32a significantly induced BMSCs to express IL-6,As the dose of rIL-32a increased(20ng/mL,40ng/mL,80ng/mL and 160ng/mL),the release of IL-6 by BMSCs achieved higher levels approximately 3 to 8 fold higher than normal.3.RPMI8226 cells induced BMSCs to express and secrete higher levels of IL-6(360.8±28.4pg/mL vs 177.3±9.4pg/mL,p<0.05).Knockdown of IL-32 in MM cells by shRNA reduced the enhancing effect toward BMSCs(246.7±10.9 pg/mL vs 360.8±28.4 pg/mL,p<0.05).Similar results were observed in OPM2 cells as well.4.Cytokine array was applied to assay more cytokines and chemokines affected by rIL-32a,not only the production of IL-6 in BMSCs,but also the expression of MIP-la and MIP-1? increased.In addition,CCL-5,IL-10 and VEGF decreased.5.rIL-32a activated NF-?B and STAT3 signaling pathways,phosphorylation of p65NF-?B and STAT3 Ser-727 increased as the dose of rIL-32a rose and the stimulation was consistent.PR3 was involved in this process,deletion of PR3 in BMSCs reduced the activation of the NF-?B and STAT3 signaling pathways and the produtction of IL-6 induced by rIL-32a.6.rIL-32a did not directly promote MM cells growth or induce MM cells apoptosis,knock down of IL-32 in MM cells also have no significant changes in proliferation or apoptosis.7.rIL-32?-pretreated BCCM significantly promoted the proliferation of H929 cells and LP-1 cells.8.In the BMSCs/MM cells co-culture system,IL-32 knock down RPMI8226 cells showed a lower proliferation rate compared to normal RPMI8226 cells,similar results were observed.in OPM2 cells as well.In vivo,IL-32 deletion in the microenvironment obviously lower tumor burdens as compared with groups of controls.9.BMSCs increase the expression of DNA damage response protein ?H2AX in MM cells,loss of IL-32 in MM cells reduced these effects.ConclusionTo summarize,our study analyzed the expression of IL-32 and its binding receptor PR3 in MM BM,and demonstrated that in the MM BM microenvironment,IL-32 regulate production of cytokines such as IL-6,CCL-5,IL-10 and VEGF in BMSCs by activating the NF-?B and STAT3 signaling pathways,thus promoting the proliferation of MM cells and inducing the genomic instability in MM cells.In expectation,IL-32 may be a new biomarker in MM diagnoses and an optimal therapy target in MM treatment.