Objective: To investigate the down-regulated TRAF6 gene expression and its effects on proliferation and apoptosis in multiple myeloma (MM) cells.
Methods: Detection of TRAF6 expression were conducted by RT-PCR and Western blot in MM cell lines of KM3, U266, RPMI8226 and primary cells from patients. RPMI8226 cell lines were transfected with siRNA of TRAF6. The efficiency of transfection was identified by using of fluorescence microscope, RT-PCR, and Western blot. The levels of proliferation were analyzed by CCK-8 method under the different concentrations of siRNA. Apoptosis rate were detected with Hoechst33258/PI double staining by flow cytometry. Apoptosis related proteins Bcl-2, BAX, and NF-κB signal pathway were observed before and after siRNA transfection by Western blot.
Results: The levels of TRAF6 mRNA and protein in MM cell lines, especially in primary myeloma cells, were significantly higher than those in controls. After transfected with 50 nmol/L siRNA in RPMI8226 cells, the relative level of TRAF6 mRNA (0.49±0.24) was significantly lower than that in non-transfected group (1.87±0.23) and idling group (1.74±0.35). The proliferation rate of siRNA transfected cells decreased with dose dependence (P<0.01). The apoptosis rates increased from 11.20% (before transfection) to 51.82% (after transfection), accompanied by down-regulated Bcl-2 protein, NF-κB signal pathway (p-p65 and p52), and up-regulated BAX protein.
Conclusion: TRAF6 expression was high in myeloma cells. TRAF6 siRNA could inhibit proliferation of myeloma cells and induce apoptosis mediated by NF-κB classical and alternative pathway in myeloma cells.