内质网蛋白质折叠分子伴侣在氟尿苷耐药绒毛膜癌JeG-3／FUDRA细胞中的作用  

Role of endoplasmic reticulum protein folding molecular chaperones in floxuridine-resistance choriocarcinoma JeG-3/FUDRA cell line

作　　者：韩冰[1] 向阳[1] 冯凤芝[1] 万希润[1] 钱海利 张雪燕 孟希亭 林晨 

机构地区：[1]中国医学科学院北京协和医学院北京协和医院妇产科,100730 [2]肿瘤研究所分子肿瘤学国家重点实验室

出　　处：《中华妇产科杂志》2013年第1期41-45,共5页Chinese Journal of Obstetrics and Gynecology

基　　金：国家自然科学基金（30973178）;国家教育部博士点基金（20101106110001）

摘　　要：目的比较氟尿苷（FUDR）耐药的绒毛膜癌（绒癌）细胞系JeG．3／FUDRA细胞和其亲本细胞系JeG一3细胞（FUDR敏感）之间表达差异的蛋白质，探讨其在绒癌耐药发生中的作用。方法采用双向差异凝胶电泳（2-DE）技术筛选JeG一3／FUDRA细胞和JeG．3细胞之间表达差异的蛋白质，基质辅助激光解吸电离飞行时间质谱（MALDI—TOF—MS）技术鉴定差异表达的蛋白质。利用靶基因功能的显著性（GO）分析和途径（Pathway）分析方法对差异表达的蛋白质进行分析和筛选，筛选出目的蛋白质。蛋白印迹法验证JeG一3／FUDRA细胞中目的蛋白质的表达；小分子干扰RNA（siRNA）干扰JeG一3／FUDRA细胞中目的蛋白质的表达后，观察其耐药性的变化。结果2-DE技术检测显示，在JeG-3／FUDRA细胞和JeG-3细胞中共筛选出46个差异表达的蛋白质，并经MALDI—TOF．MS技术鉴定证实其均为差异表达的蛋白质点；通过GO分析和Pathway分析方法对差异表达的蛋白质进行分析和筛选后发现，内质网蛋白质折叠分子伴侣钙网蛋白（CALR）、蛋白二硫异构酶A3（PDIA3）和相对分子质量为78000的葡萄糖调节蛋白（GRP78）在耐药细胞中表达均明显增高。蛋白印迹法检测显示，JeG-3／FUDRA细胞中CALR、PDIA3、GRF78蛋白的表达强度明显高于JeG-3细胞。干扰JeG一3／FUDRA细胞中CALR和PDIA3基因的表达后，JeG-3／FUDRA细胞的耐药性分别下降了76．3％（干扰前后分别为36．7±2．0和8．7±3．1，P〈0．05）和51．4％（干扰前后分别为36．7±2．0和17．8±1．2，P〈0．05）。结论内质网蛋白质折叠分子伴侣CALR、PDIA3、GRP78很可能参与了绒癌细胞FUDR耐药的发生。Objective To investigate changes of protein expression profiles between human choriocarcinoma JeG-3 cell line and its floxuridine （FUDR）-resistant sub-line. Methods The differentially expressed proteins were identified by using two dimension difference gel electrophoresis （2-DE） and matrix- assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS）approaches. Gene ontology （GO） analysis and Pathway analysis were used to screen the candidate proteins. The levels of the proteins in chemo-resistant sub-lines were validated by western blot. Ribonucleic acid interference （RNAi） was used to knockdown the expression of calreticulin （CALR） and （or） protein disulfide-isomerase A3 （PDIA3） respectively. Results Forty-six proteins spots were found to be significantly different in spot intensity by statistical analysis between chemo-resistance sub-line and parent cell line, of which 31 proteins were identified by MALDI-TOF-MS. Comparing to the parent cell lines, three endoplasmic reticulum （ER） protein folding molecular chaperones： CALR, PDIA3 and 78 000 glucose-regulated protein （GRP78） screened out were increased significantly in floxuridine-resistant sub-line and were verified by western blot. The resistance index decreased by knockdown the CALR and/or PDIA3 expression in FUDR-resistant sub-line 76. 3% （36. 7 ±2. 0 vs. 8.7 ±3.1 ,P 〈0. 05） and 51.4% （36. 7 ±2. 0 vs. 17.8 ± 1.2,P 〈0. 05） respectively. Conclusion These ER protein folding molecular chaperones, CALR, PDIA3 and GRP78 , may involved in the mechanism of FUDR-resistance choriocarcinoma.

关 键 词：绒毛膜癌 细胞系 肿瘤 抗药性 肿瘤 蛋白质折叠 氟尿苷 蛋白质二硫化物异构酶 热休克蛋白质类 

分 类 号：R737.33[医药卫生—肿瘤]