体外RNA干扰下调DEPTOR表达对人多发性骨髓瘤细胞增殖和凋亡能力的影响  

The Influence of Downregulation of DEPTOR Expression by RNA Interference on the Proliferation and Apoptosis of Human Multiple Myeloma Cells in vitro

作　　者：张浩然[1] 曾志勇[1] 陈君敏[1] 

机构地区：[1]福建医科大学附属第一医院,福州350005

出　　处：《中国生物工程杂志》2013年第5期13-21,共9页China Biotechnology

基　　金：国家自然科学基金(30871111);福建省教育厅课题(JB11056)资助项目

摘　　要：目的:研究探讨应用RNA干扰技术构建人DEPTOR基因的shRNA慢病毒载体,鉴定在多发性骨髓瘤RPMI-8226细胞上的沉默效果,并评价DEPTOR对人多发性骨髓瘤细胞增殖和凋亡能力的影响。方法:设计4个DEPTOR siRNA序列,化学合成后退火形成双链,克隆到酶切的hU6-MCS-CMV-EGFP(GV115)-shRNA慢病毒载体。重组质粒经PCR测序鉴定后,脂质体介导下转染293T细胞,Western blot检测DEPTOR的表达,筛选出干扰效果最佳的GV115-shRNA。将筛选的GV115-shRNA与慢病毒包装质粒共转染293T细胞生成病毒,收集病毒上清并浓缩,测定浓度。将病毒颗粒感染多发性骨髓瘤RPMI-8226细胞,应用Real-time PCR方法从mRNA水平及Western blot方法从蛋白水平检测DEPTOR的沉默效果。运用四甲基偶氮唑蓝(MTT)法检测骨髓瘤细胞增殖能力的变化;流式细胞仪检测细胞凋亡;用Western blot分析cleaved caspase-3和cleaved PARP的变化。结果:构建的慢病毒载体shRNA的PCR鉴定和测序正确,成功筛选出最有效抑制DEPTOR表达的shRNA2序列,包装病毒后滴度达到1×109TU/ml。慢病毒感染RPMI-8226细胞后可表达EGFP,Real-time PCR和Western blot检测DEPTOR的表达明显下降。下调DEPTOR基因可显著减慢骨髓瘤细胞的生长速度(P<0.05),肿瘤细胞的凋亡率上升(P<0.01)。DEPTOR shRNA上调cleaved caspase-3、cleaved PARP和Bax表达,下调Bcl-2及PI3K/Akt信号通路的表达(P<0.01)。结论:成功构建了DEPTOR shRNA重组慢病毒载体,其转染多发性骨髓瘤RPMI-8226细胞后显著抑制了DEPTOR的表达。DEPTOR shRNA可以有效地诱导骨髓瘤细胞的凋亡,并抑制肿瘤细胞的增殖。PI3K/Akt信号通路可能参与了其凋亡过程。Objective： To construct short hairpin RNA （shRNA） lentiviral vectors targeting human DEPTOR gene, detect its effect of gene silence and investigate the effect of DEPTOR on the proliferation and apoptosis in human multiple myeloma （MM） RPMI-8226 cells. Methods： Four human DEPTOR siRNA sequences were designed and then cloned into hU6-MCS-CMV-EGFP （GVll5）-shRNA vector. The above recombinants were transfected into 293T cells by means of lipofectamine mediation. The gene silencing efficacy of these 4 siRNA sequences was compared in Western blot analysis. The GV115-shRNA was co-transfected into 293T cells. The most effective G^115-shRNA was screened out by Western blot. GVll5-shRNA and lentiviral packaging plasmid were co-transfected into packging cell line 293T. Culture supernatants were harvested and concentrated to generate the lentivirus encoding DEPTOR-RNAi. The lentivirus particles were packaged and DEPTOR specific shRNA was transmitted into RPMI-8226 cells after screening for the valid shRNA. DEPTOR silencing efficiency was determined by Real-time PCR at mRNA level and Western blot at protein level. MTr method was used todetect the proliferation of the cells. Flow cytometry was used to detect the cell apoptosis. Changes of cleaved caspase-3 and cleaved PARP were analyzed by Western blot. Results： The constructed revealed shRNA plasmids were proved to be correct by PCR and sequencing, shRNA plasmid （GVll5-shRNA2）, which efficiently silenced DEPTOR, was screened out. GVll5-shRNA2 and lentiviral packaging plasmid were successfully packaged with a titer of 1 x 109 TU/ml. EGFP expression could be detected in RPMI-8226 after infection of the recombinant lentivirus. The expression of DEPTOR decreased obviusly detected by Real-time PCR and Western blot. Down-regulation of DEPTOR expression distinctly decrease the proliferation rates of MM line （ P 〈 0.05 ）, induce tumor cell apoptosis （P 〈 0.01 ）. DEPTOR shRNA could increase expression of cleaved caspase-3 and cleaved PARP and Bax. The

关 键 词：DEPTOR 多发性骨髓瘤RNA干扰增殖凋亡 

分 类 号：Q813[生物学—生物工程]