下调TRAF6基因表达对多发性骨髓瘤细胞增殖的影响及其机制研究  

Effects of down-regulated TRAF6 gene expression on the proliferation and apoptosis in multiple myeloma cells

作　　者：黄红铭[1] 王信峰[1] 刘新新[1] 徐瑞容[1] 丁润生[1] 姜胜华[1] 施维 

机构地区：[1]南通大学附属医院血液科,226001 [2]外科综合实验室

出　　处：《中华血液学杂志》2013年第11期941-945,共5页Chinese Journal of Hematology

基　　金：基金项目：国家自然科学基金青年科学基金（81201857）;江苏省六大人才高峰基金（2010一WS一037）

摘　　要：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Westemblot技术检测TRAF6在MM细胞系KM3、U266、RPMl8226细胞及MM原代细胞的表达；构建TRAF6小干扰RNA（siRNA），转染RPMl8226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；Wester nblot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．KB信号通路蛋白的变化。结果TRAF6mRNA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。50nmol／LsiRNA转染RPMl8226细胞后TRAF6mRNA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—wB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—KB经典和旁路途径信号传递有关。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- r,B 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- kB 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-kB classical and alternative pathway in myeloma cells.

关 键 词：多发J性骨髓瘤 基因 TRAF6 RNA干扰 

分 类 号：R733.3[医药卫生—肿瘤]