Small interfering RNA-mediated silencing of nicotinamide phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) induce growth inhibition and apoptosis in human multiple myeloma cells: A preliminary study.

Ivyna Bong Pau Ni,Shaik Kamal Fakiruddin,Ng Ching Ching,Zubaidah Zakaria,Lim Moon Nian

doi:10.17305/bjbms.2016.1568

Abstract

Multiple myeloma (MM) is a malignancy of B lymphocytes or plasma cells. Our array-based comparative genomic hybridization findings revealed chromosomal gains at 7q22.3 and 1q42.3, where nicotinamide (NAM) phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) genes are localized, respectively. This led us to further study the functions of these genes in myeloma cells. NAMPT is a key enzyme involved in nicotinamide adenine dinucleotide salvage pathway, and it is frequently overexpressed in human cancers. In contrast, little is known about the function of LYST in cancer. The expression of LYST is shown to affect lysosomal size, granule size, and autophagy in human cells. In this study, the effects of small interfering RNA (siRNA)-mediated silencing of NAMPT and LYST on cell proliferation and apoptosis were evaluated in RPMI 8226 myeloma cells. Transfection efficiencies were determined by quantitative real time reverse transcriptase PCR. Cell proliferation was determined using MTT assay, while apoptosis was analyzed with flow cytometry using Annexin V-fluorescein isothiocyanate/propidium iodide assay. The NAMPT protein expression in siRNA-treated cells was estimated by enzyme-linked immunosorbent assay. Our results showed that NAMPT and LYST were successfully knockdown by siRNA transfection (p < 0.05). NAMPT or LYST gene silencing significantly inhibited cell proliferation and induced apoptosis in RPMI 8226 cells (p < 0.05). Silencing of NAMPT gene also decreased NAMPT protein levels (p < 0.01). Our study demonstrated that NAMPT and LYST play pivotal roles in the molecular pathogenesis of MM. This is the first report describing the possible functions of LYST in myelomagenesis and its potential role as a therapeutic target in MM.

Highlights

Multiple myeloma (MM) is a malignancy of B lymphocytes or plasma cells
When small interfering RNA (siRNA) duplexes were pooled and nicotinamide phosphoribosyl transferase (NAMPT)-abc was used for the transfection, the gene knockdown efficiency was increased by up to >70% at 24 hours post-transfection (20% higher compared to the single siRNAs)
A significant decrease in the cell growth was observed in NAMPT-abc- and lysosomal trafficking regulator (LYST)-abc-treated cells when compared to the cells transfected with scrambled negative control siRNAs (p < 0.05) (Figures 3A and B). These findings showed that the suppression of NAMPT or LYST gene led to the inhibition of cell proliferation in RPMI 8226 cells

Summary

Introduction

Multiple myeloma (MM) is a malignancy of B lymphocytes or plasma cells. It is a biologically complex disease characterized by excessive numbers of abnormal plasma cells in the bone marrow, and overproduction of intact monoclonal immunoglobulin of a single type [1]. It is the second most common hematologic cancer, representing 1% of all cancer diagnoses and 2% of all cancer deaths [2]. Submitted: 28 July 2016/Accepted: 20 August 2016 patients are diagnosed at the late stage of the disease, and MM is more prevalent in men than women [3].

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