CAJ | 학술논문

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-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-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-3/FUDRA细胞中目的蛋白质的表达；小分子干扰RNA(siRNA)干扰JeG-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78 000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、GRP78蛋白的表达强度明显高于JeG-3细胞.干扰JeG-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.
목적 비교불뇨감(FUDR)내약적융모막암(융암)세포계JeG-3/FUDRA세포화기친본세포계JeG-3세포(FUDR민감)지간표체차이적단백질,탐토기재융암내약발생중적작용.방법 채용쌍향차이응효전영(2-DE)기술사선JeG-3/FUDRA세포화JeG-3세포지간표체차이적단백질,기질보조격광해흡전리비행시간질보(MALDI-TOF-MS)기술감정차이표체적단백질.이용파기인공능적현저성(GO)분석화도경(Pathway)분석방법대차이표체적단백질진행분석화사선,사선출목적단백질.단백인적법험증JeG-3/FUDRA세포중목적단백질적표체；소분자간우RNA(siRNA)간우JeG-3/FUDRA세포중목적단백질적표체후,관찰기내약성적변화.결과 2-DE기술검측현시,재JeG-3/FUDRA세포화JeG-3세포중공사선출46개차이표체적단백질,병경MALDI-TOF-MS기술감정증실기균위차이표체적단백질점；통과GO분석화Pathway분석방법대차이표체적단백질진행분석화사선후발현,내질망단백질절첩분자반려개망단백(CALR)、단백이류이구매A3(PDIA3)화상대분자질량위78 000적포도당조절단백(GRP78)재내약세포중표체균명현증고.단백인적법검측현시,JeG-3/FUDRA세포중CALR、PDIA3、GRP78단백적표체강도명현고우JeG-3세포.간우JeG-3/FUDRA세포중CALR화PDIA3기인적표체후,JeG-3/FUDRA세포적내약성분별하강료76.3％(간우전후분별위36.7±2.0화8.7±3.1,P＜0.05)화51.4％(간우전후분별위36.7±2.0화17.8±1.2,P＜0.05).결론 내질망단백질절첩분자반려CALR、PDIA3、GRP78흔가능삼여료융암세포FUDR내약적발생.
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 matrixassisted 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±2.0vs.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.