内质网蛋白质折叠及其相关通路在耐药绒癌中的作用

【作者】 韩冰；

【导师】 向阳；

【作者基本信息】 中国协和医科大学 ， 妇产科， 2010， 博士

【摘要】 研究目的：包括绒癌(CC)在内的滋养细胞肿瘤(GTN)是人类最早可以治愈的对化疗高度敏感的实体瘤,90%以上的患者可以通过规范化的化疗而治愈。但是化疗耐药仍是患者治疗失败的主要原因。迄今为止,有关CC耐药发生的确切机制尚不明确,因此,建立耐药CC的体外模型对于其耐药机制的研究尤其重要。上个世纪九十年代兴起的蛋白质组学如今正方兴未艾,它以细胞内全部蛋白质的存在以及活动方式做为研究对象,从整体的水平研究细胞、组织以及器官的蛋白质表达的变化。众所周知,肿瘤是多因素共同作用的结果,因此有必要利用蛋白质组学对耐药和敏感绒癌细胞株之间蛋白表达差异进行高通量的检测以筛查出耐药候选蛋白,从而为滋养细胞肿瘤耐药的逆转提供依据。为此,本研究采用临床滋养细胞肿瘤五种常用化疗药物(FUDR/5-FU/MTX/KSM/VP 16),使用间断和连续两种方法诱导人绒毛膜上皮癌JeG-3细胞,分别建立耐药细胞株。通过比较蛋白质组学的方法,寻找耐药株和敏感株之间蛋白表达的差异,探讨绒癌的耐药机制,为指导临床化疗方案的制定,改善GTN患者,尤其是耐药GTN患者的预后提供依据。研究方法：1.采用五种临床滋养细胞肿瘤化疗常用药物(FUDR/5-FU/MTX/KSM/VP 16),使用间断和连续两种方法诱导人绒毛膜上皮癌JeG-3细胞,分别建立相应的耐药细胞株；2.用细胞计数法检测上述各个耐药细胞株的耐药性,并比较各个细胞株的细胞形态、生长曲线、群体倍增时间和染色体倍性的差异；用化学发光法检测亲本和耐药细胞株诱导过程中β-HCG和P分泌水平的动态变化；并同时用荧光定量PCR法检测相关化疗药物作用靶基因以及常用耐药相关基因在各个耐药细胞株诱导过程中的表达变化趋势；3.利用差异凝胶电泳识别亲本和JeG-3/FUDRA1耐药细胞株差异表达的蛋白质,用MALDI-TOF-MS对差异蛋白质进行鉴定,利用生物信息学和系统生物学方法对鉴定出来的蛋白质进行筛选,确定耐药相关候选蛋白；4.用Western blot法分别验证耐药相关候选蛋白在亲本细胞株与各耐药细胞株中的表达差异,同时检测其在各个耐药细胞株诱导过程中的动态变化趋势；RNAi候选蛋白质的表达,检测转染前后细胞生物学特性和耐药性的改变；5.用细胞免疫荧光的方法,利用共聚焦显微镜检测亲本和耐药细胞以及诱导过程中候选蛋白质表达量和细胞内定位的改变；6.用荧光定量PCR的方法检测候选蛋白质基因mRNA的表达以及与候选蛋白质作用相关网络基因mRNA在亲本和耐药细胞中的表达差异。研究结果：1.历时10～14月分别建立五株间断耐药细胞株JeG-3/FUDRA1、JeG-3/FUB1、JeG-3/MTXA1、JeG-3/KSMB1和JeG-3/VPC1;三株连续耐药细胞株JeG-3/FUDRC2、JeG-3/FUC2和JeG-3/MTXC2。经无药培养基培养三月后测定其耐药指数在11.26～65.87之间,冻存半年后复苏检测其耐药性无明显回复,提示耐药性稳定；2.和亲本细胞相比,各个耐药细胞在细胞形态、生长速度、群体倍增时间、染色体倍性以及细胞周期分布等方面均有不同程度的差异；3.动态检测在药物诱导耐药过程中各个细胞p-HCG和P分泌水平变化的结果显示：随着药物诱导浓度的增加和细胞耐药性的增高,其p-HCG和P分泌呈阶段性变化,与药物诱导浓度和细胞耐药性无明显相关性；4.针对DHFR/GST-π/LRP/MDR1/MRP/survivin/TopoIIa/TS等常见耐药基因和／或相关化疗药物作用靶酶基因转录水平表达的检测结果提示：除了GST-π基因的表达与5-FU两种方式诱导以及KSM间断诱导浓度和细胞耐药性之间明显正相关、LRP基因的表达在FUDR间断和5-FU连续诱导浓度和细胞耐药性之间明显正相关,各个耐药基因和／或靶酶在相关化疗药物诱导过程中的mRNA表达水平变化与药物诱导浓度和细胞耐药性无明显相关性；5.通过差异凝胶电泳和质谱分析,在亲本JeG-3细胞株和耐药JeG-3/FUDRA1细胞株中成功鉴定出46个差异蛋白质点,得到非冗余差异蛋白质31个,其中在耐药细胞株表达上调的蛋白质15个；表达下调的蛋白质12个以及未确定蛋白质4个。分别涵盖结合、酶催化活性、结构分子活性、转录调节活性、运输、酶调节活性和抗氧化活性等七大生物学功能。其中与内质网蛋白质折叠功能相关的CALR、PDIA3和GRP78在耐药细胞株中均有上调。其上调倍数分别为1.56、2.44和1.76倍；6.用Western blot的方法在各个耐药细胞株中均检测到上述三种和内质网蛋白质折叠功能相关的蛋白质表达上调,且有随着药物诱导浓度和耐药性的增加表达逐渐上调的趋势。干扰CALR和／或PDIA3在JeG-3/FUDRA1和JeG-3/MTXA1中的表达后,细胞耐药性下降1.52～4.22倍。细胞形态无明显改变；7.共聚焦定位显示CALR、PDIA3和GRP78定位于内质网,在耐药细胞表达均较亲本细胞上调,且CALR和PDIA3蛋白在耐药细胞株诱导过程中还有细胞内定位的改变,表现为低浓度药物诱导后CALR和PDIA3呈膜聚集性表达,而高浓度药物诱导后则表现为近核内质网内表达明显增强。该定位转变的现象在VP16耐药株中尤为明显；8.与亲本细胞比较,与内质网蛋白质折叠通路相关的各个基因在各个不同化疗药物以及两种不同方式诱导的耐药株中均较亲本细胞有不同程度的表达改变。结论：1.本研究利用间断和连续两种诱导方法,历时10～14月分别成功建立了针对FUDR、5-FU、MTX、KSM和VP16的稳定耐药株；2.由于绒癌细胞分泌β-HCG的水平与药物诱导浓度和细胞耐药性之间无明显相关性,故β-HCG不适用于作为耐药绒癌的预测指标；3.就目前结果而言,GST-π和LRP基因的表达量可以作为5-FU耐药的候选标志物,同时上述两种基因转录水平的变化也可以分别预测KSM和FUDR耐药的发生；4.本研究结果显示在各个耐药细胞中三大内质网蛋白质折叠系统的主要蛋白质折叠分子伴侣CALR、PDIA3和GRP78在蛋白表达水平均明显上调,且有随药物诱导浓度和耐药性增加而逐渐上调的趋势。对CALR、PDIA3以及两个蛋白同时行RNA干扰的结果显示,下调其表达可以明显增加FUDR和MTX间断耐药株对相应药物的敏感性。提示其参与耐药的发生；5.对包括内质网未折叠蛋白反应、内质网相关降解以及内质网相关凋亡在内的内质网应激其他三大反应途径的相关基因mRNA水平的检测结果示显示：在不同化疗药物以及不同方式诱导的绒癌耐药株中均存在有不同程度的ERS和UPR激活。提示ERS相关通路有可能成为绒癌化疗耐药干涉新的靶点；6. CALR和PDIA3在亲本细胞和不同浓度药物诱导的绒癌细胞中定位的改变提示CALR和PDIA3介导的肿瘤细胞免疫源性凋亡通路异常可能参与肿瘤耐药的发生。更多还原

【Abstract】 Background:Choriocarcinoma (CC) is a form of gestational trophoblastic neoplasia (GTN). Because of its highly invasive and destructive nature, widespread metastases appear at comparatively early stages of pregnancy. Fortunately, CC is curable, and chemotherapy may be effective for 90% patients. However, drug-resistance is a main cause for the death of CC patients. Thus, understanding the mechanisms of chemo-resistance may lead to biomarkers screening, improved risk stratification and new treatment strategies.Much evidence has shown that the mechanisms responsible for chemo-resistance are multi-factorial, and analysis of a single protein as a factor determining chemo-resistance or as a biomarker of it is of limited use. Fortunately, the development of high-resolution techniques such as proteomics and progress in systems biology have made it possible to perform network investigations to identify panels of proteins involved in particular pathological conditions, as to facilitate the study of chemo-resistance mechanisms and biomarker identification.In order to clarify the mechanism of chemo-resistance of GTN, we generated chemo-resistant human CC JeG-3 sub-line with five different clinical chemo-reagents including (FUDR,5-FU, MTX, KSM and VP16) by sub-culturing JeG-3 cells with incremental and consecutive feeding methods respectively. Chemo-resistance related proteins were searched for the first time by using differential proteomics-based approaches in choriocarcinoma sensitive and resistance cell lines. Systems biology and bioinformatics were used to screen differentially expressed proteins identified by 2D-DIGE and MALDI-TOF-MS. The roles of the candidate proteins in chemo-resistance choriocarcinoma were further discussed.Methods:1. Starting from the chemo-sensitive JeG-3 human choriocarcinoma cell line, chemo-resistant variants were obtained by exposing parental cell line to stepwise increased related chemo-reagents concentrations by intermittent and consecutive feeding methods.2. The biological characteristics of all the sub-lines were examined under an inverted phase contrast microscope. IC50, cell cycle, hormone secretion and growth curve were detected in all the sub-lines. Cell Counting Kit-8 (CCK-8) assay was used to measure the inhibitory concentration 50%. qRT-PCR was used to measure dynamical profiles of DHFR, GST-π, MDR1, MRP, survivin, TopoⅡαand TS genes mRNA expression during the establishment of related chemo-resistant sub-lines.3.2D-DIGE and MALDI-TOF-MS approaches were used to identify the differential proteins in JeG-3 parent cell line and chemo-resistant sub-line. The differentially expressed proteins were screened by using systems biology and bioinformatics methods.4. The levels of the candidate proteins in chemo-resistant sub-lines were validated by western blot respectively. RNAi was used to knockdown the expression of CALR and/or PDIA3 respectively. The effects on growth and proliferation and IC50 of RNAi cells were detected.5. CALR, PDIA3 and GRP78 expression and sub-cellular location were also detected in different concentration of chemo-reagents-induced sub-lines by immunocytochemistry methods.6. qRT-PCR was used to detect the difference of the transcript levels of candidate proteins and ER function related pathway among the chemo-resistance sub-lines and parent cell line.Results:1. Five kinds of chemo-resistant sub-lines named JeG-3/FUDRA1、JeG-3/FUB1、JeG-3/MTXA1、JeG-3/KSMB1 and JeG-3/VPC1 were established by intermittent inducing method. In consecutive-inducing, three kinds of chemo-resistant sub-lines were established:JeG-3/FUDRC2、JeG-3/FUC2 and JeG-3/MTXC2. The RI of these chemo-resistant sub-lines ranged form 11.26-65.87, and had no significant change after half a year refrigeration.2. Many differences could be found among chemo-resistant sub-lines and parent JeG-3 cell line. The dynamical profile ofβ-HCG and P secretion suggested that the hormone secretions were stage-related, and had no relationship with the concentration of chemo-reagent exposure and RI.3. The transcript level of LRP seemed to be in coincidence with the concentration of FUDR-and 5-FU-exposure; the mRNA expression level of GST-πhad positive correlation with the concentration of 5-FU-and KSM-exposure and the RI. The transcription levels of other chemo-resistant related genes were all stage-related with the concentration of the drug exposure, and had no relationship with the RI.4. 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, CALR, PDIA3 and GRP78 were screened out finally. The expression folds in chemo-resistance sub-lines of these three proteins were 1.56,2.44 and 1.76 respectively.5. These three proteins were upregulated in all chemo-resistance sub-lines, and the expression levels of these three proteins seemed to enhance gradually in coincidence with the increased concentration of drug inducing and the increased RI. The RI decreased 1.52-4.22 in JeG-3/FUDRA1 and JeG-3/MTXA1 sub-lines by knockdown the CALR and/or PDIA3 expression respectively. There were no significant changes in morphology and cell growth after RNAi.6. All these three proteins were located in ER by confocal microscopy. Besides the high intensities of these three proteins were exhibited in chemo-resistance sub-lines, the translocation of CALR and PDIA3 were also found, especially in JeG-3/VPC1 sub-lines.7. The genes involved in ER protein fold-related pathway were changed to certain extention by comparing with parent cell line and chemo-resistance sub-lines.Conclusion:1. We established eight kinds of chemo-resistance choriocarcinoma sub-lines successfully.2. The disaccording profile ofβ-HCG secretion with the concentration of chemo-reagents exposure and RIs of related sub-lines indicated that the P-HCG could not be accurate for the prediction of chemo-resistance in chemo-therapy of choriocarcinoma.3. The transcript level of GST-πand LRP could serve as a reliable candidate biomarker for predicting chemo-resistance to 5-FU-based chemotherapy, and might also predict KSM-resistant and FUDR-resistant respectively.4. Three endoplasmic reticulum molecular chaperones were found to be up-regulated in all chemo-resistance sub-lines which indicated enhanced ER protein folding ability may be involved in the mechanism of chemo-resistance of GTN.5. ERS and UPR seemed to be activated in chemo-resistance choriocarcinoma cell lines, which indicated the related pathway of ER function might be the next target of chemo-resistance choriocarcinoma.6. The translocation of CALR and PDIA3 suggested that the immunogenicity of tumour cell death might participate in the mechanism of chemo-resistance.更多还原