C1QBP基因在绒毛膜癌耐药细胞株中的表达及其与耐药的关系  

Expression of C1QBP gene and its correlation with drug resistance in human resistance choriocarcinoma cell line

作　　者：沈晓燕[1] 韩冰[1] 沈芸[1] 杨隽钧[1] 任彤[1] 沙桂华[1] 向阳[1] 

机构地区：[1]中国医学科学院北京协和医院妇产科,100730

出　　处：《中华妇产科杂志》2014年第8期616-620,共5页Chinese Journal of Obstetrics and Gynecology

基　　金：国家自然科学基金（81272890）

摘　　要：目的 检测补体成分1Q亚成分结合蛋白（C1QBP）基因在绒毛膜癌（绒癌）耐药细胞株及其亲本细胞株中的表达差异,探讨以C1QBP基因为靶向的RNA干扰能否有效逆转绒癌耐药细胞株对相应化疗药物的耐药性.方法 （1）通过实时荧光定量PCR技术、蛋白印迹法和细胞免疫荧光法检测绒癌耐药细胞株,即氟脲嘧啶脱氧核苷（FUDR）耐药细胞株JeG-3/FUDR、甲氨蝶呤（MTX）耐药细胞株JeG-3/MTX、依托泊苷（VP-16）耐药细胞株JeG-3/VP、放线菌素D（ACTD,又称KSM）耐药细胞株JeG-3/KSM细胞,以及亲本细胞株JeG-3细胞中C1QBP mRNA和蛋白的表达及蛋白定位.（2）靶向构建C1QBP基因的短发夹状RNA（shRNA）,包装成C1QBP基因RNA干扰（RNAi）慢病毒表达载体,即C 1QBP-RNAi-LV,转染绒癌耐药细胞,实时荧光定量PCR技术及蛋白印迹法检测转染后绒癌耐药细胞株JeG-3/FUDR细胞和亲本细胞株JeG-3细胞中C1QBP mRNA和蛋白的表达,活细胞计数（CCK-8）法检测各绒癌耐药细胞的药物敏感性的变化.结果 （1）转染前绒癌耐药细胞株JeG-3/FUDR、JeG-3/MTX、JeG-3/VP、JeG-3/KSM细胞中C1QBP mRNA表达水平分别为2.520±0.680、1.770±0.230、1.940±0.090、1.740±0.350,均明显高于亲本细胞株JeG-3细胞（为1.000）,差异均有统计学意义（P＜0.05）.4种绒癌耐药细胞中C1QBP蛋白表达强度均高于亲本细胞株JeG-3细胞;绒癌耐药细胞和亲本细胞中均存在C1QBP蛋白的表达,均定位于细胞内的线粒体.（2）转染后绒癌耐药细胞株JeG-3/FUDR细胞中C1QBP mRNA表达水平下调了93.1％（P＜0.01）,C1QBP蛋白表达完全被抑制.（3）转染后绒癌耐药细胞株JeG-3/FUDR、JeG-3/MTX、JeG-3/VP和JeG-3/KSM细胞的耐药指数较其相应RNAi阴性对照分别下降了86.3％、93.9％、92.8％和89.9％,差异均有统计学意义（P＜0.05）.结论 C1QBP基因在绒癌耐药细胞株中表达明显增高,沉默C1QBP基因表达可以有效逆转绒癌耐药细�Objective To examine the complement component 1 Q subcomponent-binding protein （C1QBP） gene expression in human resistance choriocarcinoma cell lines and its parental cell line JeG-3,and to investigate whether silence C 1QBP by small interference RNA could reverse the resistance of human resistance choriocarcinoma cell lines to its relevant chemotherapy drugs.Methods Expression of C1QBP mRNA and protein in cells were detected by real-time fluorogenic quantitative PCR and western blot,respectively.The difference of C 1QBP expression was compared between human resistance choriocarcinoma cell lines and its parental cell line JeG-3.Sub-cellular location was proved by confocal immunofluorescence microscopy.A lentiviral vector containing short hairpin RNA （shRNA） targeting C 1QBP was constructed and cotransfected with the packaging plasmid mixture into 293T cells by lipofectamine 2000.The human resistance choriocarcinoma cell lines were infected with the packaged lentivirus.Real-time fluorogenic quantitative PCR and western blot were used to validate whether the C 1QBP gene expression was silenced.The cell counting kit 8（CCK8）was used to determine the drug sensitivity.Results （1）The C1QBP mRNA expression levels among four human resistance choriocarcinoma cell lines[JeG-3/floxuridiuum （FUDR）,JeG-3/methotrexate （MTX）,JeG-3/etoposide （VP）,JeG-3/dactinomycin （KSM）] were 2.520±0.680,1.770±0.230,1.940±0.090 and 1.740±0.350 folds compared to that in JeG-3 cells.The C1QBP protein was higher expression level in human resistance choriocarcinoma cell lines than that in JeG-3.The immunofluorescence methods and confocal analysis showed that C1QBP localized predominantly in the mitochondrial matrix.（2）The C1QBP mRNA expression in JeG-3/FUDR cells after infected with lentiviral vector were decreased by 93.1％ （P＜0.01）.The protein expression of C 1QBP in JeG-3/FUDR cells after infected with lentiviral vector were almost completely suppressed.The resistance indexes of four human resistance

关 键 词：绒毛膜癌 载体蛋白质类 线粒体蛋白质类 细胞系 肿瘤 抗药性 肿瘤 RNA干扰 

分 类 号：R737.33[医药卫生—肿瘤]