胰腺癌5-氟尿嘧啶（5-FU）耐药相关蛋白筛查及胰腺癌候选免疫原性膜抗原SLP-2和Prohibitin分子生物学功能研究
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摘要
背景
胰腺癌是消化系统中恶性程度较高、预后极差的肿瘤,其发生发展机制尚未阐明。实现早期诊断与增强化疗效果,是胰腺癌诊治过程中的重点与难点。5-氟尿嘧啶(5-fluorouracil,5-FU)是第一个用于胰腺癌化疗的药物,目前仍然广泛应用、具有一定疗效,5-FU耐药性是限制该药应用的主要困扰。国内外多项对5-FU敏感/耐药的胰腺癌或其他肿瘤细胞株的基因芯片与蛋白质组学筛查,获得了一些差异蛋白,但是重合率并不好,可能与基因芯片探针数及二维凝胶电泳所导致的检测通量不足有关。在早期诊断方面,本实验室前期工作发现了9个胰腺癌免疫相关原性膜抗原,同时已对儿茶酚胺氧位甲基转移酶(Catechol-O-Methyltransferase,COMT)、抗增殖蛋白(prohibitin,PHB)、电压依赖离子通道(Voltage-Dependent Anionchannel, VDAC1)进行了一定程度的生物功能研究与标志物有效性验证,Stomatin样蛋白2(stomatin like protein2, SLP-2,Stoml2)等其他候选蛋白的研究还有待继续深入。
目的
1.验证人胰腺癌细胞系亲本株PaTu8988/-与5-FU耐药株PaTu8988/FU对5-FU的药物敏感性差异。
2.筛查亲本株PaTu8988/-与耐药株PaTu8988/FU的差异表达基因及蛋白。
3.研究SLP-2蛋白在人胰腺癌细胞系中的生物学功能。
4.完善prohibitin蛋白过表达后所发生的细胞功能改变的研究。
材料与方法
1.应用CCK-8法检测人胰腺癌细胞系亲本株PaTu8988/-与5-FU耐药株PaTu8988/FU对5-FU的IC50差异。
2.应用Annexin V/PI双染法分析亲本株PaTu8988/-与耐药株PaTu8988/FU在5-FU诱导下发生细胞凋亡的差异。
3.应用Affymetrix人全转录组外显子芯片GeneChip(?) Exon1.0ST Array筛查亲本株PaTu8988/-与耐药株PaTu8988/FU的基因表达差异。
4.应用同位素标记相对和绝对定量技术(Isobaric tags for relative and absolute quantitation, iTRAQ)结合TripleTOFTM5600高分辨液相色谱质谱联用系统筛查亲本株PaTu8988/-与耐药株PaTu8988/FU的蛋白表达差异。
5.应用qPCR及Western blotting法检测SLP-2在9株人胰腺癌细胞系(Capan-1、Panc-1、MIAPaCa-2、T3M4、AsPC-1、BxPC-3、PaTu8988、Su86.86、 SW1990)中的表达水平。
6.构建pIERS2-EGFP(+)-SLP2过表达系统与SLP-2siRNA抑制系统,分别瞬时过表达或抑制SLP-2蛋白在细胞系MIAPaCa-2与BxPC-3中的表达水平。应用CCK-8法、Transwell法观察并对比干预前后胰腺癌细胞在增殖、迁移与侵袭能力的变化。
7.应用qPCR及Western blotting法检测prohibitin在9株人胰腺癌细胞系(Capan-1、Panc-1、MIAPaCa-2、T3M4、AsPC-1、BxPC-3、PaTu8988、Su86.86、 SW1990)中的表达水平。
8.构建pIERS2-EGFP(+)-prohibitin过表达系统,瞬时过表达prohibitin蛋白在细胞系MIAPaCa-2中的表达水平。应用CCK-8法、Transwell法观察并对比干预前后胰腺癌细胞在增殖、迁移与侵袭能力的变化。
结果
1. CCK-8法耐药敏感性检测测定两株细胞50%抑制浓度IC50, PaTu8988/-IC50=4.49±0.60μg/ml, PaTu8988/Fu IC50=79.19±21.55μg/ml,具有显著性差异(p=0.0002),耐药系数RI=17.64。
2.细胞凋亡分析(Annexin V/PI双染法)提示,在5-FU作用下,亲本株PaTu8988/-较耐药株PaTu8988/Fu更易出现细胞凋亡现象。2500μg/ml5-FU作用下,亲本株PaTu8988/-凋亡率可达45.47±2.14%,而耐药株PaTu8988/Fu凋亡率只有22.33±2.21%。
3.转录组外显子芯片筛查,发现亲本株PaTu8988/-与5-FU耐药株PaTu8988/FU差异表达mRNA2052条,其中PaTu8988/FU较PaTu8988/-上调(>2.0)1828条,下调(<0.5)224条。
4.蛋白质组学筛查,发现亲本株PaTu8988/-与5-FU耐药株PaTu8988/FU差异表达蛋白353个,其中PaTu8988/FU较PaTu8988/-上调(>1.5)259个,下调(<0.7)94个。
5.基因芯片筛查与差异蛋白质组学筛查共同筛出PaTu8988/FU较PaTu8988/-差异蛋白69个,上调蛋白ABCD3、MTHFD1L、TP53I3、POLA1、SRGAP1、C5等54个,下调蛋白EPCAM、MCAM等15个。
6. slp-2蛋白在9株人胰腺癌细胞系中均有明确表达,但表达量各有差异。结合qPCR与Western blotting结果在MIAPaCa-2中表达量较低,在BxPC-3中表达量较高,BxPC-3细胞系SLP-2表达量是MIAPaCa-2的3倍(p<0.005)。
7.在体外水平,PIERS2-EGFP(+)-SLP2过表达质粒转染72小时后,SLP-2蛋白表量可趋近高值,过表达组较对照组SLP-2蛋白表达量调高至近3倍(p<0.05),过表达组增殖、迁移、侵袭能力明显提高(p<0.005, p=0.0001, p <0.0001); SLP-2siRNA转染48小时后,SLP-2蛋白表量可趋近低限,抑制组SLP-2蛋白表达量降至对照组的1/2(p<0.05),抑制组细胞增殖、迁移、侵袭能力明显降低(p<0.05,p<0.0001,p=0.0020)。
8. Prohibitin蛋白在9株人胰腺癌细胞系中均有明确表达,但表达量各有差异。结合qPCR与Western blotting结果在MIAPaCa-2中表达量较低,在SW1990、AsPC-1、BxPC-3中表达量较高,AsPC-1细胞系prohibitin表达量是MIAPaCa-2的2-3倍(p<0.005)。
9.在体外水平,pIERS2-EGFP(+)-prohibitin过表达质粒转染72小时后,prohibitin蛋白表量可趋近高值,过表达组较对照组prohibitin蛋白表达量调高至近1.5倍(p<0.005)。过表达组增殖、迁移、侵袭能力明显提高(p<0.001,p<0.0001,p=0.0005)。
结论
1.人胰腺癌细胞系亲本株PaTu8988/与5-FU耐药株PaTu8988/FU对5-FU药物敏感性存在差异,后续耐药蛋白筛查实验有意义。
2. ABCD3、MTHFD1L、TP53I3、POLA1、SRGAP1、C5等功能蛋白以及EPCAM、MCAM等细胞连接蛋白可能是5-FU的耐药相关蛋白。
3.体外实验结果显示,SLP-2蛋白与胰腺癌细胞的增殖及迁移侵袭能力有关。
4.体外实验结果显示,prohibitin蛋白与胰腺癌细胞的增殖及迁移侵袭能力有关。
Background
Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors in gastrointestinal tract, which has the characteristics of rapid progression, low resction rate and poor prognosis. Early diagnosis and chemotherapy efficacy are2key points in PDAC medicine.5-fluorouracil (5-FU) is the first chemotherapy drug used in PDAC with the challenge of drug resistance. Some microarray and proteomic analysis was taken and some chemo-resistance related protein got detected. However, there were still no significant findings, due to lack in screening of the complete genome and proteome. In previous work, we found9immunogenic membrane antigens in PDAC, and profoundly studied the bio-function of Catechol-O-Methyl-transferase (COMT), prohibitin (PHB) and Voltage-Dependent Anionchannel (VDAC1) and their bio-marker validity. Stomatin like protein2(SLP-2, Stoml2) and other proteins are still waiting for study.
Objective
1. To identify the chemo-sensitivity difference to5-FU between pancreatic cell lines PaTu8988/-and PaTu8988/FU.
2. To screen for the differential proteins between PaTu8988/-and PaTu8988/FU.
3. To explore the bio-function of SLP-2in PDAC.
4. To complete the prohibitin study.
Methods
1. The chemo-sensitivity was detemined by CCK-8assay.
2. The cell apoptosis rate was revealed by Annexin V/PI assay.
3. The genomic difference was detected by Affymetrix GeneChip(?) Exon1.0ST Array.
4. The proteomic difference was identified by iTRAQ-TripleTOFTM5600analysis.
5. SLP-2expression in9pancreatic cell-lines was tested by qPCR and Western blotting.
6. SLP-2overexpressing plasmid, plERS2-EGFP(+)-SLP2, and specific SLP-2siRNA was transfected into MIAPaCa-2and BxPC-3separately. And the cellular ability of proliferation, migration and invasion in intro was observed.
7. Prohibitin expression in9pancreatic cell-lines was tested by qPCR and Western blotting.
8. Prohibitin overexpressing plasmid, pIERS2-EGFP(+)-prohibitin, was transfected into MIAPaCa-2. And the cellular ability of proliferation, migration and invasion in intro was observed.
Results
1. IC50of PaTu8988/FU is higher than PaTu8988/-significantly (p=0.0002), with resistance index (RI) of17.64.
2. PaTu8988/-holds a greater apoptosis rate under5-FU than PaTu8988/FU.
3. Genomic chip found2052differential mRNAs, of which1828were up-regulation and224were down-regulated in PaTu8988/FU.
4. Proteomic analysis found353differential proteins, of which259were up-regulation and94were down-regulated in PaTu8988/FU.
5. All9cell-lines expressed SLP-2protein. Higher SLP-2expression level was found in BxPC-3, otherwise MIAPaCa-2with a lower expression.
6. In vitro, pIERS2-EGFP(+)-SLP2enhanced SLP-2level in MIAPaCa-2, and also the proliferation, migration and invasion capability. SLP-2siRNA reduced SLP-2level in MIAPaCa-2, and the proliferation, migration and invasion capability as well.
7. All9cell-lines expressed prohibitin protein. Higher prohitbitin expression level was found in SW1990, AsPC-1and BxPC-3, otherwise MIAPaCa-2with a lower expression.
8. In vitro, pIERS2-EGFP(+)-prohibitin increased prohibitin level in MIAPaCa-2, and also the proliferation, migration and invasion capability.
Conclusions
1. PaTu8988/-was more sensitive to5-FU than PaTu8988/FU, offering a suitable material.
2.69differential proteins were corporately identify by both genomic chip and proteomic analysis, including54up-regulated proteins, such as ABCD3, MTHFD1L, TP53I3, POLA1, SRGAP1and C5, as well15down-regulated ones, for example EPCAM, MCAM and other cell adhesion proteins.
3. In vitro, SLP-2enhanced cell proliferation, migration and invasion.
4. In vitro, prohibitin facilitated cell proliferation, migration and invasion as well.
胰腺癌是消化系统中恶性程度较高、预后极差的肿瘤,其发生发展机制尚未阐明。实现早期诊断与增强化疗效果,是胰腺癌诊治过程中的重点与难点。5-氟尿嘧啶(5-fluorouracil,5-FU)是第一个用于胰腺癌化疗的药物,目前仍然广泛应用、具有一定疗效,5-FU耐药性是限制该药应用的主要困扰。国内外多项对5-FU敏感/耐药的胰腺癌或其他肿瘤细胞株的基因芯片与蛋白质组学筛查,获得了一些差异蛋白,但是重合率并不好,可能与基因芯片探针数及二维凝胶电泳所导致的检测通量不足有关。在早期诊断方面,本实验室前期工作发现了9个胰腺癌免疫相关原性膜抗原,同时已对儿茶酚胺氧位甲基转移酶(Catechol-O-Methyltransferase,COMT)、抗增殖蛋白(prohibitin,PHB)、电压依赖离子通道(Voltage-Dependent Anionchannel, VDAC1)进行了一定程度的生物功能研究与标志物有效性验证,Stomatin样蛋白2(stomatin like protein2, SLP-2,Stoml2)等其他候选蛋白的研究还有待继续深入。
目的
1.验证人胰腺癌细胞系亲本株PaTu8988/-与5-FU耐药株PaTu8988/FU对5-FU的药物敏感性差异。
2.筛查亲本株PaTu8988/-与耐药株PaTu8988/FU的差异表达基因及蛋白。
3.研究SLP-2蛋白在人胰腺癌细胞系中的生物学功能。
4.完善prohibitin蛋白过表达后所发生的细胞功能改变的研究。
材料与方法
1.应用CCK-8法检测人胰腺癌细胞系亲本株PaTu8988/-与5-FU耐药株PaTu8988/FU对5-FU的IC50差异。
2.应用Annexin V/PI双染法分析亲本株PaTu8988/-与耐药株PaTu8988/FU在5-FU诱导下发生细胞凋亡的差异。
3.应用Affymetrix人全转录组外显子芯片GeneChip(?) Exon1.0ST Array筛查亲本株PaTu8988/-与耐药株PaTu8988/FU的基因表达差异。
4.应用同位素标记相对和绝对定量技术(Isobaric tags for relative and absolute quantitation, iTRAQ)结合TripleTOFTM5600高分辨液相色谱质谱联用系统筛查亲本株PaTu8988/-与耐药株PaTu8988/FU的蛋白表达差异。
5.应用qPCR及Western blotting法检测SLP-2在9株人胰腺癌细胞系(Capan-1、Panc-1、MIAPaCa-2、T3M4、AsPC-1、BxPC-3、PaTu8988、Su86.86、 SW1990)中的表达水平。
6.构建pIERS2-EGFP(+)-SLP2过表达系统与SLP-2siRNA抑制系统,分别瞬时过表达或抑制SLP-2蛋白在细胞系MIAPaCa-2与BxPC-3中的表达水平。应用CCK-8法、Transwell法观察并对比干预前后胰腺癌细胞在增殖、迁移与侵袭能力的变化。
7.应用qPCR及Western blotting法检测prohibitin在9株人胰腺癌细胞系(Capan-1、Panc-1、MIAPaCa-2、T3M4、AsPC-1、BxPC-3、PaTu8988、Su86.86、 SW1990)中的表达水平。
8.构建pIERS2-EGFP(+)-prohibitin过表达系统,瞬时过表达prohibitin蛋白在细胞系MIAPaCa-2中的表达水平。应用CCK-8法、Transwell法观察并对比干预前后胰腺癌细胞在增殖、迁移与侵袭能力的变化。
结果
1. CCK-8法耐药敏感性检测测定两株细胞50%抑制浓度IC50, PaTu8988/-IC50=4.49±0.60μg/ml, PaTu8988/Fu IC50=79.19±21.55μg/ml,具有显著性差异(p=0.0002),耐药系数RI=17.64。
2.细胞凋亡分析(Annexin V/PI双染法)提示,在5-FU作用下,亲本株PaTu8988/-较耐药株PaTu8988/Fu更易出现细胞凋亡现象。2500μg/ml5-FU作用下,亲本株PaTu8988/-凋亡率可达45.47±2.14%,而耐药株PaTu8988/Fu凋亡率只有22.33±2.21%。
3.转录组外显子芯片筛查,发现亲本株PaTu8988/-与5-FU耐药株PaTu8988/FU差异表达mRNA2052条,其中PaTu8988/FU较PaTu8988/-上调(>2.0)1828条,下调(<0.5)224条。
4.蛋白质组学筛查,发现亲本株PaTu8988/-与5-FU耐药株PaTu8988/FU差异表达蛋白353个,其中PaTu8988/FU较PaTu8988/-上调(>1.5)259个,下调(<0.7)94个。
5.基因芯片筛查与差异蛋白质组学筛查共同筛出PaTu8988/FU较PaTu8988/-差异蛋白69个,上调蛋白ABCD3、MTHFD1L、TP53I3、POLA1、SRGAP1、C5等54个,下调蛋白EPCAM、MCAM等15个。
6. slp-2蛋白在9株人胰腺癌细胞系中均有明确表达,但表达量各有差异。结合qPCR与Western blotting结果在MIAPaCa-2中表达量较低,在BxPC-3中表达量较高,BxPC-3细胞系SLP-2表达量是MIAPaCa-2的3倍(p<0.005)。
7.在体外水平,PIERS2-EGFP(+)-SLP2过表达质粒转染72小时后,SLP-2蛋白表量可趋近高值,过表达组较对照组SLP-2蛋白表达量调高至近3倍(p<0.05),过表达组增殖、迁移、侵袭能力明显提高(p<0.005, p=0.0001, p <0.0001); SLP-2siRNA转染48小时后,SLP-2蛋白表量可趋近低限,抑制组SLP-2蛋白表达量降至对照组的1/2(p<0.05),抑制组细胞增殖、迁移、侵袭能力明显降低(p<0.05,p<0.0001,p=0.0020)。
8. Prohibitin蛋白在9株人胰腺癌细胞系中均有明确表达,但表达量各有差异。结合qPCR与Western blotting结果在MIAPaCa-2中表达量较低,在SW1990、AsPC-1、BxPC-3中表达量较高,AsPC-1细胞系prohibitin表达量是MIAPaCa-2的2-3倍(p<0.005)。
9.在体外水平,pIERS2-EGFP(+)-prohibitin过表达质粒转染72小时后,prohibitin蛋白表量可趋近高值,过表达组较对照组prohibitin蛋白表达量调高至近1.5倍(p<0.005)。过表达组增殖、迁移、侵袭能力明显提高(p<0.001,p<0.0001,p=0.0005)。
结论
1.人胰腺癌细胞系亲本株PaTu8988/与5-FU耐药株PaTu8988/FU对5-FU药物敏感性存在差异,后续耐药蛋白筛查实验有意义。
2. ABCD3、MTHFD1L、TP53I3、POLA1、SRGAP1、C5等功能蛋白以及EPCAM、MCAM等细胞连接蛋白可能是5-FU的耐药相关蛋白。
3.体外实验结果显示,SLP-2蛋白与胰腺癌细胞的增殖及迁移侵袭能力有关。
4.体外实验结果显示,prohibitin蛋白与胰腺癌细胞的增殖及迁移侵袭能力有关。
Background
Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors in gastrointestinal tract, which has the characteristics of rapid progression, low resction rate and poor prognosis. Early diagnosis and chemotherapy efficacy are2key points in PDAC medicine.5-fluorouracil (5-FU) is the first chemotherapy drug used in PDAC with the challenge of drug resistance. Some microarray and proteomic analysis was taken and some chemo-resistance related protein got detected. However, there were still no significant findings, due to lack in screening of the complete genome and proteome. In previous work, we found9immunogenic membrane antigens in PDAC, and profoundly studied the bio-function of Catechol-O-Methyl-transferase (COMT), prohibitin (PHB) and Voltage-Dependent Anionchannel (VDAC1) and their bio-marker validity. Stomatin like protein2(SLP-2, Stoml2) and other proteins are still waiting for study.
Objective
1. To identify the chemo-sensitivity difference to5-FU between pancreatic cell lines PaTu8988/-and PaTu8988/FU.
2. To screen for the differential proteins between PaTu8988/-and PaTu8988/FU.
3. To explore the bio-function of SLP-2in PDAC.
4. To complete the prohibitin study.
Methods
1. The chemo-sensitivity was detemined by CCK-8assay.
2. The cell apoptosis rate was revealed by Annexin V/PI assay.
3. The genomic difference was detected by Affymetrix GeneChip(?) Exon1.0ST Array.
4. The proteomic difference was identified by iTRAQ-TripleTOFTM5600analysis.
5. SLP-2expression in9pancreatic cell-lines was tested by qPCR and Western blotting.
6. SLP-2overexpressing plasmid, plERS2-EGFP(+)-SLP2, and specific SLP-2siRNA was transfected into MIAPaCa-2and BxPC-3separately. And the cellular ability of proliferation, migration and invasion in intro was observed.
7. Prohibitin expression in9pancreatic cell-lines was tested by qPCR and Western blotting.
8. Prohibitin overexpressing plasmid, pIERS2-EGFP(+)-prohibitin, was transfected into MIAPaCa-2. And the cellular ability of proliferation, migration and invasion in intro was observed.
Results
1. IC50of PaTu8988/FU is higher than PaTu8988/-significantly (p=0.0002), with resistance index (RI) of17.64.
2. PaTu8988/-holds a greater apoptosis rate under5-FU than PaTu8988/FU.
3. Genomic chip found2052differential mRNAs, of which1828were up-regulation and224were down-regulated in PaTu8988/FU.
4. Proteomic analysis found353differential proteins, of which259were up-regulation and94were down-regulated in PaTu8988/FU.
5. All9cell-lines expressed SLP-2protein. Higher SLP-2expression level was found in BxPC-3, otherwise MIAPaCa-2with a lower expression.
6. In vitro, pIERS2-EGFP(+)-SLP2enhanced SLP-2level in MIAPaCa-2, and also the proliferation, migration and invasion capability. SLP-2siRNA reduced SLP-2level in MIAPaCa-2, and the proliferation, migration and invasion capability as well.
7. All9cell-lines expressed prohibitin protein. Higher prohitbitin expression level was found in SW1990, AsPC-1and BxPC-3, otherwise MIAPaCa-2with a lower expression.
8. In vitro, pIERS2-EGFP(+)-prohibitin increased prohibitin level in MIAPaCa-2, and also the proliferation, migration and invasion capability.
Conclusions
1. PaTu8988/-was more sensitive to5-FU than PaTu8988/FU, offering a suitable material.
2.69differential proteins were corporately identify by both genomic chip and proteomic analysis, including54up-regulated proteins, such as ABCD3, MTHFD1L, TP53I3, POLA1, SRGAP1and C5, as well15down-regulated ones, for example EPCAM, MCAM and other cell adhesion proteins.
3. In vitro, SLP-2enhanced cell proliferation, migration and invasion.
4. In vitro, prohibitin facilitated cell proliferation, migration and invasion as well.
引文
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6. Maehara S, Tanaka S, Shimada M, et al. Selenoprotein P, as a predictor for evaluating gemcitabine resistance in human pancreatic cancer cells. Int. J. Cancer: 2004;112(2):184-9.
7.陈革,赵玉沛,郭俊超,et al.胰腺癌细胞株耐药与凋亡相关基因的改变.外科理论与实践.2004；9(4)：325-8.
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9. Zhao YP, Chen G, Feng B, et al. Microarray analysis of gene expression profile of multidrug resistance in pancreatic cancer. Chinese Medical Journal. 2007;120(20):1743-52.
10. Yoshida K, Kuramitsu Y, Murakami K, et al. Proteomic differential display analysis for TS-1-resistant and -sensitive pancreatic cancer cells using two-dimensional gel electrophoresis and mass spectrometry. Anticancer Research.2011;31(6):2103-8.
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18. Xue A, Scarlett CJ, Chung L, et al. Discovery of serum biomarkers for pancreatic adenocarcinoma using proteomic analysis. British Journal of Cancer. 2010;103(3):391-400.
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