MRP1参与粘液表皮样癌多药耐药机制的研究
发布时间：2018-09-19 06:24
【摘要】：无论是西方世界还是中国，粘液表皮样癌都是口腔以及头颈部最常见的原发性唾液腺肿瘤[1,2,3]。粘液表皮样癌占唾液腺所有肿瘤的10%，占唾液腺恶性肿瘤的35%[4]。在儿童患者中，粘液表皮样癌更是占到了唾液腺所有肿瘤的16%，占唾液腺恶性肿瘤的51%[5]。根据肿瘤形态和肿瘤细胞特征，粘液表皮样癌分为高度恶性粘液表皮样癌，中度恶性粘液表皮样癌，低度恶性粘液表皮样癌。中度恶性粘液表皮样癌和低度恶性粘液表皮样癌一般情况下生存率很高，然而高度恶性粘液表皮样癌一般情况下预后很差，其五年生存率仅有30%左右[6,7]。粘液表皮样癌通常对化疗及放疗均不敏感，手术切除一直是粘液表皮样癌的标准治疗方法，化疗仅用于无法完全切除的粘液表皮样癌患者或者已经远端转移的粘液表皮样癌患者，即便是这样，粘液表皮样癌患者的化疗预后仍然不甚理想[8]，并且头颈部的肿瘤切除总会给患者带来难以恢复的头颈部疤痕和面部缺陷，极大的影响了患者的生活质量并给患者带来极大的心理创伤。因此提高粘液表皮样癌的化疗疗效成为当务之急。本研究的目的就是通过研究粘液表皮样癌的耐药机制，找出粘液表皮样癌耐药的原因，从而为日后针对粘液表皮样癌的治疗打下坚实的基础。 多药耐药相关蛋白（MPR1或ABCC1）一直以来被认为是一个膜结合，能量依赖的蛋白转运子。隶属于ABC转运通道蛋白家族。多药耐药蛋白（MDR1或ABCB1）被首次发现后，所有人都认为找到了治疗肿瘤耐药的关键分子，认为多药耐药蛋白MDR1就是引起肿瘤耐药的唯一转运蛋白。然而，在一个多药耐药小细胞肺癌细胞系中[9]却发现多药耐药蛋白（MDR1或ABCB1）没有过表达，过表达的是另一种转运蛋白—多药耐药相关蛋白MRP1，这是MRP1的首次发现。之后，大量报道发现MRP1的过表达可以预测多种血液和实体瘤化疗的不良预后[10]。在肿瘤细胞中，尽管MRP1也被发现与细胞质中，但MRP1主要位于细胞膜上也表明将药物从细胞内泵到细胞外是MRP1导致多药耐药中的主要原因[11,12,13,14,15,16]。在正常组织中，MRP1主要位于细胞基底外侧面，用来将底物排入血液中[17]。MDR1和MRP1都属于ATP结合蛋白超家族成员，并且都是和多药耐药高度相关的蛋白。人类MDR1在染色体7q21上，，跨度100kb；人类MRP1在染色体16p13.1上，跨度为194kb，两基因氨基酸序列相似度仅为15%。MDR1底物通常为中性疏水药物或带阳性电荷的疏水药物，而MRP1的底物通常为中性疏水药物或带阴性电荷的疏水药物。与MDR1不同的是，MRP1也可以转运谷胱甘肽及谷胱甘肽的药物结合物[18]。虽然对MRP1的研究很多，但是MRP1的晶体结构和它的转运机制仍然不甚明了[19]，并且肿瘤中的MRP1由于其广泛存在基因多态性和变异，使得MRP1的功能更加难以预测[20,21]. 本次研究中，我们在粘液表皮样癌细胞中首次发现了核MRP1的存在，并证明了MRP1的核转运和粘液表皮样癌的多药耐药的相关性。为研究MRP1在粘液表皮样癌细胞中是如何发挥多药耐药作用的，我们利用RNA干扰技术下调MRP1表达，并发现MDR1的表达也相应的下降了。我们通过免疫组化技术检测了MDR1和MRP1在127例粘液表皮样癌患者肿瘤样本中的的蛋白表达，也发现二者之间的正相关关系。通过免疫组化检测组织芯片，发现MRP1的核表达并未在其他正常组织和肿瘤组织中出现。为了证明MDR1的下调确实是由MRP1的下调引起，我们通过荧光素酶报告分析技术最终证明了MRP1下调后，通过改变MDR1基因启动子的活性，从而引起MDR1的表达量改变。综上所述，我们认为MRP1的新功能可能为将来粘液表皮样癌的临床个体化治疗提供新的靶点。 第一部分MRP1下调引起粘液表皮样癌多药耐药细胞系耐药性下降 目的：研究MRP1在粘液表皮样癌多药耐药中所发挥的作用。 材料和方法:高转移粘液表皮样癌细胞系MC3和由其使用五氟尿嘧啶冲击疗法诱导而出的多药耐药细胞系MC3/5FU由本实验室建系并保存。合成针对MRP1的shRNA，并筛选出被其稳定转染的MC3/5FU细胞系。通过逆转录聚合酶链反应(RT-PCR)来检测RNA的表达，蛋白质印迹（Western blot）用来检测蛋白表达的改变。甲基噻唑基四唑试验(MTT assay)用来检测细胞生长和细胞耐药性的改变。末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法（TUNEL staining）用来检测细胞凋亡。数据结果使用Student’s t-test and one-way ANOVA (LSD)来计算对比实验组与对照组之间的差异显著性。计算由统计软件SPSS version12.0完成。P0.05为有显著差异。结果: MRP1的下调显著提高了多药耐药细胞系MC3/5FU的对五氟尿嘧啶(5FU)，阿霉素(ADM)，表阿霉素(PADM)，博来霉素-A5(BLM-A5)，顺铂(CDDP)和紫杉醇(TAX)的化学敏感性，在一定5FU浓度下，MRP1的下调显著抑制了MC3/5FU细胞的生长并显著增加了其凋亡。 结论:MRP1在粘液表皮样癌的多药耐药中发挥着重要作用。 第二部分. MRP1的核转位参与粘液表皮样癌的多药耐药性改变 目的：检测MRP1的核转移是否与粘液表皮样癌的多药耐药性的改变相关。材料和方法:免疫荧光细胞化学染色法进行细胞染色后，使用激光共聚焦显微镜用来检测MRP1的亚细胞分布。免疫荧光组织化学法来检测MRP1在粘液表皮样癌肿瘤组织中和正常腮腺组织中的表达以及定位，并通过免疫化学组织染色法来对组织芯片染色，从而检测MRP1在其他正常组织和其他肿瘤组织中的表达和定位。Student’st-test and one-way ANOVA (LSD)用来计算对比改变的显著性以及实验组和对照组之间的差异。计算由统计软件SPSS版本12.0完成，P0.05为有显著差异。 结果:通过聚合酶链反应和免疫印迹的方法，在RNA以及蛋白水平，对比MRP1在多药耐药细胞系MC3/5FU中和其亲代细胞MC3中的表达，我们发现MRP1的表达并没有发生量的变化。但是当我们下调MRP1在MCA/5FU细胞中的表达后，细胞的多药耐药性却明显下降了。免疫荧光细胞化学染色进行蛋白定位后发现MRP1从MC3细胞的细胞膜及细胞质中转移到了MC3/5FU细胞的细胞核中，而且MC3/5FU细胞中下调的MRP1主要为细胞核中的MRP1。为检测核MRP1是否也出现在其他组织或者肿瘤中，也为了检测是否为MRP1的一抗的非特异性染色才使得MRP1出现在细胞核中，我们用相同的一抗对组织芯片在相同条件下进行免疫化学染色，结果我们并未发现MRP1在其他正常组织或者肿瘤组织中有核表达。 结论: MRP1的核转移参与粘液表皮样癌的多药耐药性改变。MRP1的核转移可能是MRP1引发耐药的新机制，基于这个新机制可能开发出新的粘液表皮样癌治疗方法。核MRP1可能为粘液表皮样癌所特有，其有潜质来成为鉴别诊断粘液表皮样癌的一个Marker。 第三部分下调MRP1的表达导致MDR1表达的下降 目的:研究核MRP1是通过何种机制来参与粘液表皮样癌的多药耐药的。 材料和方法:从第四军医大学口腔医院病理科收集127例常规手术治疗粘液表皮样癌患者的肿瘤组织样本,患者手术时间均在2006年至2011年。用免疫组织化学染色来检测MRP1和MDR1在粘液表皮样癌组织中的的表达。染色程度由蔡卜磊博士和刘园博士分别检测。结果通过定性的方法结合定量的方法检测。为排除MRP1表达在细胞核膜上的可能性，利用激光共聚焦显微镜进行分层扫描进行蛋白定位。 RT-PCR用来检测RNA表达量的改变。Western blot用来检测蛋白表达的改变。Student’s t-test andone-way ANOVA (LSD)用来计算对比改变的显著性。斯皮尔曼等级相关分析来分析MDR1表达量和MRP1表达量的相关性。计算由统计软件SPSS version12.0完成，P0.05为有显著差异。 结果:为了排除MRP1一抗特异性的问题，我们从另一家公司购买了新的MRP1一抗来进行免疫组织化学染色，结果我们再次确认MRP1出现在粘液表皮样癌的细胞核中。聚合酶链反应以及免疫印迹法检测发现，在下调MRP1的表达后，MDR1的表达也明显降低。通过分析127例粘液表皮样癌患者的肿瘤样本相同位置的MRP1表达和MDR1表达及定位，再次确认发现MRP1的表达量与MDR1的表达量有明显正相关关系。 结论:在粘液表皮样癌肿瘤中MRP1的表达量与MDR1的表达量显著正相关。 第四部分核MRP1通过降低MDR1启动子的活性来调节MDR1的表达 目的:初步探索MRP1对MDR1表达通路中的影响 材料和方法: shRNA瞬时转染MC3/5FU细胞系下调MRP1的表达。逆转录聚合酶链反应用来检测RNA的表达，免疫印迹法用来检测蛋白表达的改变。荧光素酶报告分析（luciferase reporter assays）检测MDR1启动子活性。Student’s t-test and one-wayANOVA (LSD)用来计算对比改变的显著性。计算由统计软件SPSS version12.0完成，P0.05为有显著差异。 结果:瞬时转染shRNA后，MRP1表达显著下调，同时荧光素酶报告分析发现MDR1启动子活性显著降低。 结论:核MRP1通过降低MDR1启动子的活性来调节MDR1的表达，其具体调控机制仍需我们进一步研究。
[Abstract]:Mucoepidermoid carcinoma is the most common primary salivary gland tumor in the oral cavity and head and neck, accounting for 10% of all salivary gland tumors and 35% of all salivary gland malignancies. According to tumor morphology and tumor cell characteristics, mucoepidermoid carcinoma can be divided into highly malignant mucoepidermoid carcinoma, moderately malignant mucoepidermoid carcinoma, and low malignant mucoepidermoid carcinoma. Epidermal carcinomas usually have a poor prognosis, with a 5-year survival rate of only about 30%[6,7]. Mucoepidermoid carcinomas are usually insensitive to chemotherapy and radiotherapy. Surgical resection has always been the standard treatment for mucoepidermoid carcinomas. Chemotherapy is used only in patients with unresectable mucoepidermoid carcinomas or mucoepidermoid carcinomas with distal metastasis. Even so, the prognosis of patients with mucoepidermoid carcinoma after chemotherapy is still not satisfactory [8], and the removal of head and neck tumors will always bring difficult to recover the head and neck scars and facial defects, greatly affecting the quality of life of patients and bring great psychological trauma to patients. The aim of this study is to find out the reason of drug resistance of mucoepidermoid carcinoma by studying the mechanism of drug resistance of mucoepidermoid carcinoma, so as to lay a solid foundation for future treatment of mucoepidermoid carcinoma.
Multidrug resistance-associated proteins (MPR1 or ABCC1) have long been considered to be membrane-bound, energy-dependent protein transporters. They belong to the ABC transporter family. After the first discovery of multidrug resistance proteins (MDR1 or ABCB1), all believed that a key molecule for the treatment of cancer resistance was found and that multidrug resistance protein MDR1 was the cause. However, multidrug resistance protein (MDR1 or ABCB1) was not overexpressed in a multidrug-resistant small cell lung cancer cell line [9], and another transporter, multidrug resistance-associated protein MRP1, was overexpressed. This was the first discovery of MRP1. Subsequently, a large number of reports found that the overexpression of MRP1 could predict more. Poor prognosis of chemotherapy in blood and solid tumors [10]. In tumor cells, although MRP1 is also found in the cytoplasm, the presence of MRP1 on the cell membrane also suggests that drug pumping from the cell to the cell is the main cause of multidrug resistance [11, 12, 13, 14, 15, 16]. In normal tissues, MRP1 is mainly located in the lateral basal surface of the cell. Both MDR1 and MRP1 belong to the ATP-binding protein superfamily and are highly multidrug-resistant proteins. Unlike MDR1, MRP1 also transports glutathione and glutathione drug conjugates [18]. Although much research has been done on MRP1, the crystal structure and transport mechanism of MRP1 remain unclear. [19], and MRP1 in tumors is more difficult to predict because of its widespread genetic polymorphisms and variations [20,21].
In this study, we first found the presence of nuclear MRP1 in mucoepidermoid carcinoma cells and demonstrated the correlation between nuclear transport of MRP1 and multidrug resistance in mucoepidermoid carcinoma. The expression of MDR1 and MRP1 in tumor samples from 127 patients with mucoepidermoid carcinoma was detected by immunohistochemistry, and a positive correlation was found between them. In order to prove that the down-regulation of MDR1 is indeed caused by the down-regulation of MRP1, we have finally demonstrated that the down-regulation of MRP1 can alter the expression of MDR1 by altering the activity of the promoter of MDR1 gene. Treatment provides new targets.
Part one, downregulation of MRP1 results in decreased drug resistance of MDR cell lines in mucoepidermoid carcinoma.
Objective: To study the role of MRP1 in the multidrug resistance of mucoepidermoid carcinoma.
Materials and Methods: High metastatic mucoepidermoid carcinoma cell line MC3 and multidrug resistant cell line MC3/5FU induced by pentafluorouracil were established and preserved in our laboratory. shRNA targeting MRP1 was synthesized and stable transfected MC3/5FU cell line was screened. RNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Methylthiazolyl tetrazolium assay (MTT assay) was used to detect cell growth and changes in cell resistance. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL staining) was used to detect apoptosis. Data were obtained using Student Results Results: The downregulation of MRP1 significantly increased the levels of pentafluorouracil (5FU), adriamycin (ADM), epirubicin (PADM), bleomycin (Bleomycin) and adriamycin (ADM) in multidrug resistant cell lines MC3/5FU. Chemosensitivity of BLM-A5, CDDP and TAX, the down-regulation of MRP1 significantly inhibited the growth and increased the apoptosis of MC3/5FU cells at a certain concentration of 5FU.
Conclusion: MRP1 plays an important role in the multidrug resistance of mucoepidermoid carcinoma.
The second part. The nuclear translocation of MRP1 is involved in the change of multidrug resistance in mucoepidermoid carcinoma.
AIM: To investigate whether nuclear metastasis of MRP1 is associated with multidrug resistance in mucoepidermoid carcinoma. Materials and Methods: Immunofluorescence cytochemical staining was used to detect the subcellular distribution of MRP1 after cell staining, and immunofluorescence histochemical staining was used to detect MRP1 in mucoepidermoid carcinoma. The expression and localization of MRP1 in normal and normal parotid gland tissues were detected by immunohistochemical staining. Student's st-test and one-way ANOVA (LSD) were used to calculate the significance of the contrast changes and to compare the difference between the experimental group and the control group. The difference is calculated by statistical software SPSS version 12. P0.05 has significant differences.
RESULTS: By polymerase chain reaction and Western blot, we compared the expression of MRP1 in multidrug resistant cell line MC3/5FU and its parental cells MC3 at RNA and protein levels, and found that the expression of MRP1 did not change quantitatively. Immunofluorescent cytochemical staining showed that MRP1 was transferred from the membrane and cytoplasm of MC3 cells to the nucleus of MC3/5FU cells, and the down-regulated MRP1 in MC3/5FU cells was mainly MRP1 in the nucleus. Whether it is the non-specific staining of the first antibody to MRP1 that causes MRP1 to appear in the nucleus, we used the same first antibody to immunostain the tissue chip under the same conditions, and we did not find that MRP1 was expressed in other normal tissues or tumor tissues.
Conclusion: The nuclear metastasis of MRP1 may be involved in the change of multidrug resistance in mucoepidermoid carcinoma. The nuclear metastasis of MRP1 may be a new mechanism of drug resistance induced by MRP1. Based on this new mechanism, a new therapy for mucoepidermoid carcinoma may be developed. A Marker.
The third part downregulated the expression of MRP1, resulting in the decrease of MDR1 expression.
Objective: To investigate the mechanism by which nuclear MRP1 can participate in multidrug resistance of mucoepidermoid carcinoma.
Materials and Methods: 127 specimens of mucoepidermoid carcinoma were collected from the Department of Pathology, Stomatology Hospital, Fourth Military Medical University. The operation time was from 2006 to 2011. The expression of MRP1 and MDR1 in mucoepidermoid carcinoma was detected by immunohistochemical staining. Results Qualitative and quantitative methods were used to detect the expression of MRP1. In order to exclude the possibility of MRP1 expression on the nuclear membrane, laser confocal microscopy was used to locate the protein. RT-PCR was used to detect the change of RNA expression. Western blot was used to detect the change of protein expression. Estandone-way ANOVA (LSD) was used to calculate the significance of contrast changes. Spearman rank correlation analysis was used to analyze the correlation between MDR1 expression and MRP1 expression.
RESULTS: In order to eliminate the specificity of MRP1 antibody, we purchased a new MRP1 antibody from another company for immunohistochemical staining. As a result, we reconfirmed the presence of MRP1 in the nucleus of mucoepidermoid carcinoma. By analyzing the expression and localization of MRP1 and MDR1 in the same location of tumor samples from 127 patients with mucoepidermoid carcinoma, it was confirmed that the expression of MRP1 was positively correlated with the expression of MDR1.
Conclusion: the expression of MRP1 is positively correlated with the expression of MDR1 in mucoepidermoid carcinoma.
The fourth part of the nuclear MRP1 regulates the expression of MDR1 by decreasing the activity of MDR1 promoter.
Objective: To explore the effect of MRP1 on MDR1 expression pathway.
Materials and Methods: The expression of MRP1 was down-regulated by shRNA transfection in MC3/5FU cell line. Reverse transcription polymerase chain reaction was used to detect the expression of RNA and Western blotting was used to detect the change of protein expression. Luciferase reporter assays were used to detect the activity of MDR1 promoter. Student's t-test and one-way ANOVA (LSD) were used to measure the activity of MDR1 promoter. The significance of the change was calculated. The calculation was completed by the statistical software SPSS version12.0, and P0.05 was significantly different.
Results: The expression of MRP1 was significantly down-regulated after shRNA transfection, and the activity of MDR1 promoter was significantly decreased by Luciferase Report analysis.
CONCLUSION: Nuclear MRP1 regulates the expression of MDR1 by decreasing the activity of MDR1 promoter, and its specific regulatory mechanism needs further study.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.87
本文编号：2249310
[Abstract]:Mucoepidermoid carcinoma is the most common primary salivary gland tumor in the oral cavity and head and neck, accounting for 10% of all salivary gland tumors and 35% of all salivary gland malignancies. According to tumor morphology and tumor cell characteristics, mucoepidermoid carcinoma can be divided into highly malignant mucoepidermoid carcinoma, moderately malignant mucoepidermoid carcinoma, and low malignant mucoepidermoid carcinoma. Epidermal carcinomas usually have a poor prognosis, with a 5-year survival rate of only about 30%[6,7]. Mucoepidermoid carcinomas are usually insensitive to chemotherapy and radiotherapy. Surgical resection has always been the standard treatment for mucoepidermoid carcinomas. Chemotherapy is used only in patients with unresectable mucoepidermoid carcinomas or mucoepidermoid carcinomas with distal metastasis. Even so, the prognosis of patients with mucoepidermoid carcinoma after chemotherapy is still not satisfactory [8], and the removal of head and neck tumors will always bring difficult to recover the head and neck scars and facial defects, greatly affecting the quality of life of patients and bring great psychological trauma to patients. The aim of this study is to find out the reason of drug resistance of mucoepidermoid carcinoma by studying the mechanism of drug resistance of mucoepidermoid carcinoma, so as to lay a solid foundation for future treatment of mucoepidermoid carcinoma.
Multidrug resistance-associated proteins (MPR1 or ABCC1) have long been considered to be membrane-bound, energy-dependent protein transporters. They belong to the ABC transporter family. After the first discovery of multidrug resistance proteins (MDR1 or ABCB1), all believed that a key molecule for the treatment of cancer resistance was found and that multidrug resistance protein MDR1 was the cause. However, multidrug resistance protein (MDR1 or ABCB1) was not overexpressed in a multidrug-resistant small cell lung cancer cell line [9], and another transporter, multidrug resistance-associated protein MRP1, was overexpressed. This was the first discovery of MRP1. Subsequently, a large number of reports found that the overexpression of MRP1 could predict more. Poor prognosis of chemotherapy in blood and solid tumors [10]. In tumor cells, although MRP1 is also found in the cytoplasm, the presence of MRP1 on the cell membrane also suggests that drug pumping from the cell to the cell is the main cause of multidrug resistance [11, 12, 13, 14, 15, 16]. In normal tissues, MRP1 is mainly located in the lateral basal surface of the cell. Both MDR1 and MRP1 belong to the ATP-binding protein superfamily and are highly multidrug-resistant proteins. Unlike MDR1, MRP1 also transports glutathione and glutathione drug conjugates [18]. Although much research has been done on MRP1, the crystal structure and transport mechanism of MRP1 remain unclear. [19], and MRP1 in tumors is more difficult to predict because of its widespread genetic polymorphisms and variations [20,21].
In this study, we first found the presence of nuclear MRP1 in mucoepidermoid carcinoma cells and demonstrated the correlation between nuclear transport of MRP1 and multidrug resistance in mucoepidermoid carcinoma. The expression of MDR1 and MRP1 in tumor samples from 127 patients with mucoepidermoid carcinoma was detected by immunohistochemistry, and a positive correlation was found between them. In order to prove that the down-regulation of MDR1 is indeed caused by the down-regulation of MRP1, we have finally demonstrated that the down-regulation of MRP1 can alter the expression of MDR1 by altering the activity of the promoter of MDR1 gene. Treatment provides new targets.
Part one, downregulation of MRP1 results in decreased drug resistance of MDR cell lines in mucoepidermoid carcinoma.
Objective: To study the role of MRP1 in the multidrug resistance of mucoepidermoid carcinoma.
Materials and Methods: High metastatic mucoepidermoid carcinoma cell line MC3 and multidrug resistant cell line MC3/5FU induced by pentafluorouracil were established and preserved in our laboratory. shRNA targeting MRP1 was synthesized and stable transfected MC3/5FU cell line was screened. RNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Methylthiazolyl tetrazolium assay (MTT assay) was used to detect cell growth and changes in cell resistance. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL staining) was used to detect apoptosis. Data were obtained using Student Results Results: The downregulation of MRP1 significantly increased the levels of pentafluorouracil (5FU), adriamycin (ADM), epirubicin (PADM), bleomycin (Bleomycin) and adriamycin (ADM) in multidrug resistant cell lines MC3/5FU. Chemosensitivity of BLM-A5, CDDP and TAX, the down-regulation of MRP1 significantly inhibited the growth and increased the apoptosis of MC3/5FU cells at a certain concentration of 5FU.
Conclusion: MRP1 plays an important role in the multidrug resistance of mucoepidermoid carcinoma.
The second part. The nuclear translocation of MRP1 is involved in the change of multidrug resistance in mucoepidermoid carcinoma.
AIM: To investigate whether nuclear metastasis of MRP1 is associated with multidrug resistance in mucoepidermoid carcinoma. Materials and Methods: Immunofluorescence cytochemical staining was used to detect the subcellular distribution of MRP1 after cell staining, and immunofluorescence histochemical staining was used to detect MRP1 in mucoepidermoid carcinoma. The expression and localization of MRP1 in normal and normal parotid gland tissues were detected by immunohistochemical staining. Student's st-test and one-way ANOVA (LSD) were used to calculate the significance of the contrast changes and to compare the difference between the experimental group and the control group. The difference is calculated by statistical software SPSS version 12. P0.05 has significant differences.
RESULTS: By polymerase chain reaction and Western blot, we compared the expression of MRP1 in multidrug resistant cell line MC3/5FU and its parental cells MC3 at RNA and protein levels, and found that the expression of MRP1 did not change quantitatively. Immunofluorescent cytochemical staining showed that MRP1 was transferred from the membrane and cytoplasm of MC3 cells to the nucleus of MC3/5FU cells, and the down-regulated MRP1 in MC3/5FU cells was mainly MRP1 in the nucleus. Whether it is the non-specific staining of the first antibody to MRP1 that causes MRP1 to appear in the nucleus, we used the same first antibody to immunostain the tissue chip under the same conditions, and we did not find that MRP1 was expressed in other normal tissues or tumor tissues.
Conclusion: The nuclear metastasis of MRP1 may be involved in the change of multidrug resistance in mucoepidermoid carcinoma. The nuclear metastasis of MRP1 may be a new mechanism of drug resistance induced by MRP1. Based on this new mechanism, a new therapy for mucoepidermoid carcinoma may be developed. A Marker.
The third part downregulated the expression of MRP1, resulting in the decrease of MDR1 expression.
Objective: To investigate the mechanism by which nuclear MRP1 can participate in multidrug resistance of mucoepidermoid carcinoma.
Materials and Methods: 127 specimens of mucoepidermoid carcinoma were collected from the Department of Pathology, Stomatology Hospital, Fourth Military Medical University. The operation time was from 2006 to 2011. The expression of MRP1 and MDR1 in mucoepidermoid carcinoma was detected by immunohistochemical staining. Results Qualitative and quantitative methods were used to detect the expression of MRP1. In order to exclude the possibility of MRP1 expression on the nuclear membrane, laser confocal microscopy was used to locate the protein. RT-PCR was used to detect the change of RNA expression. Western blot was used to detect the change of protein expression. Estandone-way ANOVA (LSD) was used to calculate the significance of contrast changes. Spearman rank correlation analysis was used to analyze the correlation between MDR1 expression and MRP1 expression.
RESULTS: In order to eliminate the specificity of MRP1 antibody, we purchased a new MRP1 antibody from another company for immunohistochemical staining. As a result, we reconfirmed the presence of MRP1 in the nucleus of mucoepidermoid carcinoma. By analyzing the expression and localization of MRP1 and MDR1 in the same location of tumor samples from 127 patients with mucoepidermoid carcinoma, it was confirmed that the expression of MRP1 was positively correlated with the expression of MDR1.
Conclusion: the expression of MRP1 is positively correlated with the expression of MDR1 in mucoepidermoid carcinoma.
The fourth part of the nuclear MRP1 regulates the expression of MDR1 by decreasing the activity of MDR1 promoter.
Objective: To explore the effect of MRP1 on MDR1 expression pathway.
Materials and Methods: The expression of MRP1 was down-regulated by shRNA transfection in MC3/5FU cell line. Reverse transcription polymerase chain reaction was used to detect the expression of RNA and Western blotting was used to detect the change of protein expression. Luciferase reporter assays were used to detect the activity of MDR1 promoter. Student's t-test and one-way ANOVA (LSD) were used to measure the activity of MDR1 promoter. The significance of the change was calculated. The calculation was completed by the statistical software SPSS version12.0, and P0.05 was significantly different.
Results: The expression of MRP1 was significantly down-regulated after shRNA transfection, and the activity of MDR1 promoter was significantly decreased by Luciferase Report analysis.
CONCLUSION: Nuclear MRP1 regulates the expression of MDR1 by decreasing the activity of MDR1 promoter, and its specific regulatory mechanism needs further study.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.87
【参考文献】
相关期刊论文 前1条
1 温玉明,代晓明,王昌美,李龙江,付风华,王晓毅,唐休发,刘华,华成舸,潘剑;口腔颌面部恶性肿瘤6539例临床病理分析[J];华西口腔医学杂志;2001年05期
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