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Interleukin-32α Promotes the Proliferation of Multiple Myeloma Cells by Inducing Production of IL-6 in Bone Marrow Stromal Cells

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Interleukin-32α Promotes the Proliferation of Multiple Myeloma Cells by Inducing Production of IL-6 in Bone Marrow Stromal Cells

Xuanru Lin et al. Oncotarget.

Abstract

Multiple myeloma (MM) is a malignant plasma disease closely associated with inflammation. In MM bone marrow microenvironment, bone marrow stromal cells (BMSCs) are the primary source of interleukin-6 (IL-6) secretion, which promotes the proliferation and progression of MM cells. However, it is still unknown how the microenvironment stimulates BMSCs to secrete IL-6. Interleukin-32 (IL-32) is a newly identified pro-inflammatory factor. It was reported that in solid tumors, IL-32 induces changes in other inflammatory factors including IL-6, IL-10, and TNF-α. The aim of this study was to investigate the expression of IL-32 and the role of IL-32 in the MM bone marrow microenvironment. Our data illustrate that MM patients have higher expression of IL-32 than healthy individuals in both bone marrow and peripheral blood. We used ELISA and qRT-PCR to find that malignant plasma cells are the primary source of IL-32 production in MM bone marrow. ELISA and Western blot analysis revealed that recombinant IL-32α induces production of IL-6 in BMSCs by activating NF-κB and STAT3 signaling pathways, konckdown of IL-32 receptor PR3 inhibit this process. Knockdown of IL-32 by shRNA decreased the proliferation in MM cells that induced by BMSCs. In conclusion, IL-32 secreted from MM cells has paracrine effect to induce production of IL-6 in BMSCs, thus feedback to promote MM cells growth.

Keywords: IL-6; bone marrow stromal cells; interleukin-32; multiple myeloma.

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no competing financial interest.

Figures

Figure 1
Figure 1. Expression of IL-32 in MM patients, human myeloma cells and BMSCs
(A) Expression of IL-32 in BM and PB, MM patients are compared to normal healthy donors (CTR), measured by ELISA. (B) Immunofluorescence analysis of MM patients BM biopsies with anti-CD138, anti-IL-32 antibodies (magnification: ×400). (C) PCR analysis of the expression of IL-32 in RPMI8226, OPM2 and BMSCs. BMSCs obtained from different MM patient (n=9). (D) Expression of soluble IL-32 in RPMI8226, OPM2 and BMSCs, measured by ELISA; BMSCs obtained from different MM patient (n=9). (E) PCR analysis of the expression of IL-32 in MM cell lines. * p<0.05.
Figure 2
Figure 2. Expression of IL-32 in primary MM cells
(A) qRT-PCR analysis of the expression of IL-32 in primary CD138+ and CD138 cells isolated from MM patients. (B) Primary CD138+ and CD138 cells isolated from MM patients were cultured in vitro for 12h (inclusion criteria: cell apoptosis<50%, detected by flow cytometry), ELISA was applied to detect soluble IL-32 in the culture medium. (C) qRT-PCR analysis of the expression of different IL-32 isoforms (IL-32α, IL-32β, IL-32γ) in primary CD138+ and CD138 cells isolated from MM patients. * p<0.05.
Figure 3
Figure 3. Cytokine array analysis in BMSCs induced by rIL-32α
(A) Cytokines and chemokines changes in BMSCs with or without 40 ng/mL rIL-32α stimulation. These images show the results of BMSCs obtained from one patient. (B) (C) Total analysis of cytokines and chemokines in BMSCs obtained from different patients (n=4).
Figure 4
Figure 4. IL-32α induces production of IL-6 in MM BMSCs
(A) Concentration of IL-6 in BMSCs in the presence of 0-160 ng/mL rIL-32α, stimulated for 24, 48, and 72 h. BMSCs obtained from one MM patient, repeated in three independent experiments, measured by ELISA. (B) Concentration of TNF-α in BMSCs in the presence of 0-320 ng/mL rIL-32α, stimulated for 24, 48 and 72 h. Samples obtained from one MM patient, repeated in three independent experiments, measured by ELISA. (C) Concentration of IL-6 in BMSCs in the presence of 0-40 ng/mL rIL-32α, stimulated for 24 h, BMSCs obtained from different patients, and 6 in 9 (66.7%) showed positive effects, measured by ELISA. (D) Concentration of IL-6 in BMSCs co-cultured with IL-32 high-expression MM cell lines, with or without IL-32 knockdown; BMSCs were co-cultured with MM cells in a 24-well Transwell plate, repeated in three independent experiments, measured by ELISA. (E) Cell proliferation of BMSCs in the presence of 0-80 ng/mL rIL-32α, stimulated for 1, 3, 5 and 7 d; cells were cultured in 96 well plates, repeated in three independent experiments, measured by CCK8 assay. (F) Identification of IL-32 knockdown MM cell lines, Western blot analysis.
Figure 5
Figure 5. IL-32α activates the NF-κB and STAT3 signaling pathways in BMSCs
(A) Western blot analysisof p38, NF-κB and STAT3 signaling pathway and in BMSCs with or without 40 ng/mL rIL-32α stimulation for 60 min. (B) Western blot analysisof NF-κB and STAT3 signaling pathwaysin BMSCs with 20-40 ng/mL rIL-32α stimulation for 60 min. (C) Western blot analysisof NF-κB and STAT3 signaling pathwaysin BMSCs with 40 ng/mL rIL-32α stimulation for 60-120 min. (D) Concentration of IL-6 in BMSCs in the presence of 40 ng/mL rIL-32α for 24 h, with or without NF-κB inhibitor QNZ (10μM/mL), STAT3 inhibitor BP-1-102 (10μM/mL), measured by ELISA; BMSCs obtained from one MM patient, repeated in three independent experiments. * p<0.05.
Figure 6
Figure 6. PR3 involved in IL-32 induced IL-6 production in BMSCs
(A) qRT-PCR analysis of the expression of PR3 in BMSCs and MM cells. (B) Concentration of IL-6 in BMSCs in the presence of 40 ng/mL rIL-32α for 24 h, with or without PR3 knockdown, BMSCs obtained from one MM patient, repeated in three independent experiments, measured by ELISA. (C) Identification of PR3 knockdown BMSCs. (D) Western blot analysisof NF-κB and STAT3 signaling pathwaysin BMSCs in the presence of 20 ng/mL rIL-32α for 60min, with or without PR3 knockdown. * p<0.05.
Figure 7
Figure 7. IL-32α promotes the proliferation of MM cells in the tumor microenvironment
(A) Cell proliferation of MM cell lines during exposure to 40 ng/mL rIL-32α for 24 h. Repeated in three independent experiments, measured by CCK8. (B) (C) Cell proliferation in IL-32 low-expression MM cell lines, H929 and LP-1, cultured in BCCM stimulated by 0-80 ng/mL rIL-32α for 24, 48 and 72 h. MM cells were cultured in 96-well plates. Repeated in three independent experiments, measured by CCK8. (D) (E) Cell proliferation in IL-32 high-expression MM cell lines, RPMI8226 and OPM2, cultured alone or co-cultured with BMSCs for 24, 48, and 72 h, with or without IL-32 knockdown. rIL-32α rescue concentration was 40ng/mL. MM cells were co-cultured with BMSCs in 24-well plates and transferred to 96-well plates to be assayed. Repeated in three independent experiments, measured by CCK8. (F) Cell proliferation in IL-6 dependent MM cell line ANBL-6, cultured in BCCM stimulated by 0-80 ng/mL rIL-32α for 24, 48 and 72 h, with or without IL-6 neutralization antibodies (20ng/mL); cells in control group were cultured in normal medium with rIL-6 (20ng/mL); MM cells were cultured in 96-well plates. Repeated in three independent experiments, measured by CCK8.
Figure 8
Figure 8. The proposed mechanism underlying the observed pro-inflammation effect of IL-32
IL-32 secreted from MM cells, increases IL-6 expression in BMSCs through activating NF-κB and STAT3 inflammation signaling pathways, thus feedback to support MM cells, resulting in the proliferation and growth of MM cells.

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