Yonsei Med J.  2018 Jan;59(1):43-50. 10.3349/ymj.2018.59.1.43.

TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling

• Chen Y
• Zhao J
• Li D
• Hao J
• He P
• Wang H
• Zhang M

• KMID: 2418847
• DOI: http://doi.org/10.3349/ymj.2018.59.1.43

Abstract

PURPOSE
Tripartite-motif-containing protein 56 (TRIM56) has been found to exhibit a broad antiviral activity, depending upon E3 ligase activity. Here, we attempted to evaluate the function of TRIM56 in multiple myeloma (MM) and its underlying molecular basis.
MATERIALS AND METHODS
TRIM56 expression at the mRNA and protein level was measured by qRT PCR and western blot analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry analysis was performed to investigate the effect of TRIM56 on MM cell proliferation and apoptosis. The concentrations of interferon (IFN)-β, interleukin (IL)-6, and tumor necrosis factor-α in MM cell culture supernatants were detected with respective commercial ELISA kits. Western blot was employed to determine the effect of TRIM56 on toll-like receptor 3 (TLR3)/toll-IL-1 receptor (TIR) domain-containing adaptor inducing IFN-β (TRIF) signaling pathway.
RESULTS
TRIM56 expression was prominently decreased in MM cells. Poly (dA:dT)-induced TRIM56 overexpression in U266 cells suppressed proliferation, induced apoptosis, and enhanced inflammatory cytokine production, while TRIM56 knockdown improved growth, diminished apoptosis, and inhibited inflammatory cytokine secretion in RPMI8226 cells. Moreover, TRIM56 knockdown blocked TLR3 signaling pathway. Furthermore, poly (I:C), a TLR3 agonist, markedly abolished TRIM56 depletion-induced increase of proliferation, decrease of apoptosis, and reduction of inflammatory factor in MM cells.
CONCLUSION
TRIM56 may act as a tumor suppressor in MM through activation of TLR3/TRIF signaling pathway, contributing to a better understanding of the molecular mechanism of TRIM56 involvement in MM pathogenesis and providing a promising therapy strategy for patients with MM.

Keyword

TRIM56; multiple myeloma; proliferation; apoptosis; inflammatory cytokine; TLR3/TRIF

MeSH Terms

Adaptor Proteins, Vesicular Transport/*metabolism
Apoptosis/drug effects
Cell Line, Tumor
Cell Proliferation/drug effects
Cytokines/secretion
*Disease Progression
Down-Regulation/drug effects
Gene Knockdown Techniques
Humans
Multiple Myeloma/*metabolism/*pathology
Poly I-C/pharmacology
*Signal Transduction/drug effects
Toll-Like Receptor 3/*metabolism
Tripartite Motif Proteins/deficiency/*metabolism
Ubiquitin-Protein Ligases/deficiency/*metabolism
Adaptor Proteins, Vesicular Transport
Cytokines
Toll-Like Receptor 3
Tripartite Motif Proteins
Ubiquitin-Protein Ligases

Figure

• 

  Fig. 1 Down-regulation of TRIM56 in MM cells. (A and B) qRT-PCR and western blot was performed to examine the expression level of TRIM56 mRNA and protein in normal PCs and different MM cell lines (NCI-H929, RPMI8226, LP-1, OPM2, and U266 cells). *p<0.05. TRIM56, tripartite-mortif-containing protein 56; MM, multiple myeloma; PC, plasma cell.

• 

  Fig. 2 TRIM56 inhibited MM cell proliferation and induced apoptosis. Western blot analysis of TRIM56 level in U266 cells (A) transfected with poly (dA:dT) and in RPMI8226 cells (E) introduced with si-control, T56si-1, or T56si-2. Cell viability was evaluated by MTT assay in U266 cells (B) with poly (dA:dT) stimulation and in RPMI8226 cells (F) transfected with si-control or T56si-2. Apoptosis detection was performed by flow cytometry analysis in U266 cells (C) with poly (dA:dT) transfection and in 2 µM ATO-treated RPMI8226 cells (G) with si-control or T56si-2 transfection. The relative caspase3 activity was measured by the Caspase3 Colorimetric Assay Kit in U266 cells (D) with poly (dA:dT) transfection and in 2 µM ATO-treated RPMI8226 cells (H) introduced with si-control or T56si-2. *p<0.05. TRIM56, tripartite-mortif-containing protein 56; MM, multiple myeloma; ATO, arsenic trioxide.

• 

  Fig. 3 TRIM56 enhanced the secretion of cytokines in MM cells. ELISA was conducted to measure the concentrations of IFN-β (A and D), IL-6 (B and E), and TNF-α (C and F) in poly (dA:dT)-treated U266 cells and T56si-2-transfected RPMI8229 cells. *p<0.05. TRIM56, tripartite-mortif-containing protein 56; MM, multiple myeloma; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

• 

  Fig. 4 TRIM56 knockdown inactivated TLR3/TRIF signaling pathway in MM cells. (A) Western blot analysis of TLR3, TRIF, and RIP1 protein in RPMI8226 cells transfected with T56si-2 or si-control. (B) Quantitative analysis of TLR3, TRIF, and RIP1 protein levels. *p<0.05. TRIM56, tripartite-mortif-containing protein 56; MM, multiple myeloma; TLR3, toll-like receptor3; TRIF, toll-IL-1 receptor domain-containing adaptor inducing interferon-β; RIP1, receptor-interacting protein 1.

• 

  Fig. 5 TRIM56 deficiency promoted the progression of MM via interrupting TLR3/TRIF signaling pathway. RPMI8226 cells were transfected with T56si-2 or along with poly (I:C) for further analysis at 48 h post-transfection. (A) Cell viability of transfected RPMI8226 cells was assessed by MTT assay. (B) Apoptotic rate of transfected RPMI8226 cells was detected by flow cytometry. (C) The relative caspase3 activity of transfected RPMI8226 cells was measured by the Caspase3 Colorimetric Assay Kit. The concentrations of secreted cytokines IFN-β (D), IL-6 (E), and TNF-α (F) in transfected RPMI8226 cells were measured by ELISA. (G) Western blot analysis of TLR3, TRIF, and RIP1 protein expression in transfected RPMI8226 cells. *p<0.05. TRIM56, tripartite-mortif-containing protein 56; MM, multiple myeloma; TLR3, toll-like receptor3; TRIF, toll-IL-1 receptor domain-containing adaptor inducing IFN-β; RIP1, receptor-interacting protein 1; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; ATO, arsenic trioxide.

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