Visfatin promotes multiple myeloma cell proliferation and inhibits apoptosis by inducing IL-6 production via NF-κB pathways

Abstract

Background: Multiple myeloma (MM) is the second most prevalent hematological malignancy that results in the proliferation of malignant plasma cells and the overproduction of monoclonal immunoglobulin. Visfatin plays an important role in the regulation of apoptosis, oxidative stress, and inflammation; however, to this date, the role of visfatin in multiple myeloma is unclear.

Objective: To explore the role of visfatin in multiple myeloma and find new targets for MM treatment.

Methods: In this study, expression of visfatin in bone marrow was detected by ELISA. The diagnostic value of visfatin was determined by receiver operating characteristic (ROC) curve analysis. After the quality control by performing western blot to confirm the knockdown of visfatin in two MM cell lines, the phenotype (proliferation and apoptosis) of visfatin in MM was determined by carrying out in vitro experiments, including CCK8, flow cytometry, and western blot. Several cytokines were determined by real-time PCR, followed by in vivo experiments and immunohistochemical assays. IκB, NF-κbp65, and phosphorylation were determined by western blot.

Results: We found that visfatin level increased in the bone marrow of MM patients compared to controls. ROC curve analysis result showed that bone marrow visfatin was able to distinguish MM patients from controls. In vitro and in vivo, visfatin promotes MM cell proliferation. The production of IL-6 was attenuated by visfatin knockdown. Furthermore, we showed that visfatin could activate IL-6 production via the NF-κB signaling pathway.

Conclusions: In MM, visfatin promotes tumor progression by upregulating IL-6 production, which may be a novel therapeutic target for the treatment of MM patients.

Keywords: Apoptosis; Interleukin-6; Multiple myeloma; Nuclear factor kappa-B; Visfatin.

Fig.1

Nampt (the gene name of visfatin) is overexpressed in MM. A Heatmap representation of differential gene expression between MM and healthy controls. B Volcano Plot was used to visualize differentially expressed genes between MM and healthy controls. C Expression of Nampt in NPC, MGUS, SMM and MM. NPC: normal plamsa cells, MGUS: monoclonal gammopathy of undermined significance, SMM: smoldering multiple myeloma. *P < 0.05, **P < 0.01, ***P < 0.001

Fig.2

Diagnostic value of visfatin in MM patients. A Visfatin levels in bone marrow serum from MM patients and controls. Wilcoxon rank-sum test was used to analyze. B ROC analysis was performed to evaluate the performance of visfatin in distinguishing MM from controls. Correlation between visfatin and cytokines and BMI. C–H Correlation between visfatin and IL-6, IL-8, IL-2, IL-10, IL-17, and BMI. Correlation analyses were performed using Spearman correlation. *P < 0.05

Fig.3

Visfatin regulates the proliferation and apoptosis of MM cells. A The CCK8 assay showed that visfatin regulated cell proliferation in AMO-1 and RPMI-8226 cell lines. Analysis of one-way ANOVA was performed for comparisons among multiple groups. B Flow cytometry to detect apoptosis rate in two cell lines treated with visfatin (100 ng/ml). Student’s t-test was used to analyze. *P < 0.05, **P < 0.01

Fig. 4

Knockdown of visfatin regulates the proliferation of MM cells. A Visfatin expression in different MM cell lines. B The knockdown efficiency of visfatin in AMO-1 and RPMI-8826 cells. C For the AMO cell line, sh-visfatin1was choosen, and for the RPMI-8226 cell line, sh-visfatin3 was choosen. D The OD values of the two cell lines were detected by CCK8. Student’s t-test was used to analyze. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Knockdown of visfatin regulates the apoptosis of MM cells. A Flow cytometry to detect apoptosis rate in the sh-visfatin and sh-NC groups. Student’s t-test was used to analyze. B Western blot assay to detect the expression of BAX, BCL2, Caspase3, Caspase9 and visfatin. Student’s t-test was used to analyze.*P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Visfatin regulates the expression of cytokines in MM cells. A The mRNA expression of IL-6, IL-8, IL-17, IL-32, and TGF-β in AMO-1 and RMI-8226 cell lines was tested by qRT-PCR. Student’s t-test was used to analyze. B The knockdown efficiency of visfatin and the expression of IL-6 in AMO-1 and RPMI-8826 cells were checked by western blot analysis. Student’s t-test was used to analyze. *P < 0.05, **P < 0.01

Fig.7

Signaling pathway involved in IL-6 production is regulated by visfatin in MM cells. Wilcoxon rank-sum test was used to analyze. A The knockdown efficiency of visfatin in AMO-1 and RPMI-8826 cells. B Phosphorylation IκB, IκB, phosphorylation NF-κB p65 subunit, and NF-κB p65 subunit were detected by western blot. C IL-6, phosphorylation IκB, IκB, phosphorylation NF-κB p65 subunit, and NF-κB p65 subunit in AMO-1 and RPMI-8226 cell lines treated with betulinic acid (BA) was measured by western blot. *P < 0.05

Fig. 8

Visfatin promotes MM cell proliferation in vivo. Student’s t-test was used to analyze. A The size of xenograft tumors in each group of mice, the weight and volume of the transplanted tumors. B IHC staining of Ki67 and IL-6. *P < 0.05, **P < 0.01

Fig. 9

A schematic model for visfatin stimulated IL-6 production in MM cells