CAJ | 학술논문

目的探讨下调肿瘤坏死因子受体相关因子6(TRAF6)表达对多发性骨髓瘤(MM)细胞增殖的影响及其机制.方法采用RT-PCR及Western blot技术检测TRAF6在MM细胞系KM3、U266、RPMI8226细胞及MM原代细胞的表达；构建TRAF6小干扰RNA (siRNA),转染RPMI8226细胞,荧光显微镜观察、RT-PCR和Western blot检测转染效率,CCK-8法测定不同浓度siRNA沉默TRAF6表达后细胞增殖情况,Hoechst33258/碘化丙锭(PI)双染法分析细胞凋亡率；Western blot法观察沉默TRAF6基因表达前后凋亡相关蛋白Bcl-2、BAX表达情况和下游NF-κB信号通路蛋白的变化.结果 TRAF6 mRNA和蛋白表达水平在MM细胞系和MM原代细胞均明显增高,尤以MM原代细胞为甚.50 nmol/L siRNA转染RPMI8226细胞后TRAF6 mRNA相对表达水平(0.49±0.24)明显低于未转染组(1.87±0.23)及阴性对照转染组(1.74±0.35).siRNA干扰后细胞增殖受抑,且呈剂量依赖性.细胞凋亡率由11.20％上升到51.82％,同时Bcl-2蛋白表达下调、BAX表达升高,NF-κB通路蛋白p-p65、p52的活性下降.结论 TRAF6在骨髓瘤细胞中表达增高,下调TRAF6表达可明显抑制骨髓瘤细胞增殖并诱导其凋亡,其作用可能与影响骨髓瘤细胞的NF-κB经典和旁路途径信号传递有关.
목적 탐토하조종류배사인자수체상관인자6(TRAF6)표체대다발성골수류(MM)세포증식적영향급기궤제.방법 채용RT-PCR급Western blot기술검측TRAF6재MM세포계KM3、U266、RPMI8226세포급MM원대세포적표체；구건TRAF6소간우RNA (siRNA),전염RPMI8226세포,형광현미경관찰、RT-PCR화Western blot검측전염효솔,CCK-8법측정불동농도siRNA침묵TRAF6표체후세포증식정황,Hoechst33258/전화병정(PI)쌍염법분석세포조망솔；Western blot법관찰침묵TRAF6기인표체전후조망상관단백Bcl-2、BAX표체정황화하유NF-κB신호통로단백적변화.결과 TRAF6 mRNA화단백표체수평재MM세포계화MM원대세포균명현증고,우이MM원대세포위심.50 nmol/L siRNA전염RPMI8226세포후TRAF6 mRNA상대표체수평(0.49±0.24)명현저우미전염조(1.87±0.23)급음성대조전염조(1.74±0.35).siRNA간우후세포증식수억,차정제량의뢰성.세포조망솔유11.20％상승도51.82％,동시Bcl-2단백표체하조、BAX표체승고,NF-κB통로단백p-p65、p52적활성하강.결론 TRAF6재골수류세포중표체증고,하조TRAF6표체가명현억제골수류세포증식병유도기조망,기작용가능여영향골수류세포적NF-κB경전화방로도경신호전체유관.
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.