MRP1參與粘液表皮樣癌多藥耐藥機(jī)制的研究
發(fā)布時(shí)間：2018-09-19 06:24
【摘要】：無論是西方世界還是中國，粘液表皮樣癌都是口腔以及頭頸部最常見的原發(fā)性唾液腺腫瘤[1,2,3]。粘液表皮樣癌占唾液腺所有腫瘤的10%，占唾液腺惡性腫瘤的35%[4]。在兒童患者中，粘液表皮樣癌更是占到了唾液腺所有腫瘤的16%，占唾液腺惡性腫瘤的51%[5]。根據(jù)腫瘤形態(tài)和腫瘤細(xì)胞特征，粘液表皮樣癌分為高度惡性粘液表皮樣癌，中度惡性粘液表皮樣癌，低度惡性粘液表皮樣癌。中度惡性粘液表皮樣癌和低度惡性粘液表皮樣癌一般情況下生存率很高，然而高度惡性粘液表皮樣癌一般情況下預(yù)后很差，其五年生存率僅有30%左右[6,7]。粘液表皮樣癌通常對化療及放療均不敏感，手術(shù)切除一直是粘液表皮樣癌的標(biāo)準(zhǔn)治療方法，化療僅用于無法完全切除的粘液表皮樣癌患者或者已經(jīng)遠(yuǎn)端轉(zhuǎn)移的粘液表皮樣癌患者，即便是這樣，粘液表皮樣癌患者的化療預(yù)后仍然不甚理想[8]，并且頭頸部的腫瘤切除總會(huì)給患者帶來難以恢復(fù)的頭頸部疤痕和面部缺陷，極大的影響了患者的生活質(zhì)量并給患者帶來極大的心理創(chuàng)傷。因此提高粘液表皮樣癌的化療療效成為當(dāng)務(wù)之急。本研究的目的就是通過研究粘液表皮樣癌的耐藥機(jī)制，找出粘液表皮樣癌耐藥的原因，從而為日后針對粘液表皮樣癌的治療打下堅(jiān)實(shí)的基礎(chǔ)。 多藥耐藥相關(guān)蛋白（MPR1或ABCC1）一直以來被認(rèn)為是一個(gè)膜結(jié)合，能量依賴的蛋白轉(zhuǎn)運(yùn)子。隸屬于ABC轉(zhuǎn)運(yùn)通道蛋白家族。多藥耐藥蛋白（MDR1或ABCB1）被首次發(fā)現(xiàn)后，所有人都認(rèn)為找到了治療腫瘤耐藥的關(guān)鍵分子，認(rèn)為多藥耐藥蛋白MDR1就是引起腫瘤耐藥的唯一轉(zhuǎn)運(yùn)蛋白。然而，在一個(gè)多藥耐藥小細(xì)胞肺癌細(xì)胞系中[9]卻發(fā)現(xiàn)多藥耐藥蛋白（MDR1或ABCB1）沒有過表達(dá)，過表達(dá)的是另一種轉(zhuǎn)運(yùn)蛋白—多藥耐藥相關(guān)蛋白MRP1，這是MRP1的首次發(fā)現(xiàn)。之后，大量報(bào)道發(fā)現(xiàn)MRP1的過表達(dá)可以預(yù)測多種血液和實(shí)體瘤化療的不良預(yù)后[10]。在腫瘤細(xì)胞中，盡管MRP1也被發(fā)現(xiàn)與細(xì)胞質(zhì)中，但MRP1主要位于細(xì)胞膜上也表明將藥物從細(xì)胞內(nèi)泵到細(xì)胞外是MRP1導(dǎo)致多藥耐藥中的主要原因[11,12,13,14,15,16]。在正常組織中，MRP1主要位于細(xì)胞基底外側(cè)面，用來將底物排入血液中[17]。MDR1和MRP1都屬于ATP結(jié)合蛋白超家族成員，并且都是和多藥耐藥高度相關(guān)的蛋白。人類MDR1在染色體7q21上，，跨度100kb；人類MRP1在染色體16p13.1上，跨度為194kb，兩基因氨基酸序列相似度僅為15%。MDR1底物通常為中性疏水藥物或帶陽性電荷的疏水藥物，而MRP1的底物通常為中性疏水藥物或帶陰性電荷的疏水藥物。與MDR1不同的是，MRP1也可以轉(zhuǎn)運(yùn)谷胱甘肽及谷胱甘肽的藥物結(jié)合物[18]。雖然對MRP1的研究很多，但是MRP1的晶體結(jié)構(gòu)和它的轉(zhuǎn)運(yùn)機(jī)制仍然不甚明了[19]，并且腫瘤中的MRP1由于其廣泛存在基因多態(tài)性和變異，使得MRP1的功能更加難以預(yù)測[20,21]. 本次研究中，我們在粘液表皮樣癌細(xì)胞中首次發(fā)現(xiàn)了核MRP1的存在，并證明了MRP1的核轉(zhuǎn)運(yùn)和粘液表皮樣癌的多藥耐藥的相關(guān)性。為研究MRP1在粘液表皮樣癌細(xì)胞中是如何發(fā)揮多藥耐藥作用的，我們利用RNA干擾技術(shù)下調(diào)MRP1表達(dá)，并發(fā)現(xiàn)MDR1的表達(dá)也相應(yīng)的下降了。我們通過免疫組化技術(shù)檢測了MDR1和MRP1在127例粘液表皮樣癌患者腫瘤樣本中的的蛋白表達(dá)，也發(fā)現(xiàn)二者之間的正相關(guān)關(guān)系。通過免疫組化檢測組織芯片，發(fā)現(xiàn)MRP1的核表達(dá)并未在其他正常組織和腫瘤組織中出現(xiàn)。為了證明MDR1的下調(diào)確實(shí)是由MRP1的下調(diào)引起，我們通過熒光素酶報(bào)告分析技術(shù)最終證明了MRP1下調(diào)后，通過改變MDR1基因啟動(dòng)子的活性，從而引起MDR1的表達(dá)量改變。綜上所述，我們認(rèn)為MRP1的新功能可能為將來粘液表皮樣癌的臨床個(gè)體化治療提供新的靶點(diǎn)。 第一部分MRP1下調(diào)引起粘液表皮樣癌多藥耐藥細(xì)胞系耐藥性下降 目的：研究MRP1在粘液表皮樣癌多藥耐藥中所發(fā)揮的作用。 材料和方法:高轉(zhuǎn)移粘液表皮樣癌細(xì)胞系MC3和由其使用五氟尿嘧啶沖擊療法誘導(dǎo)而出的多藥耐藥細(xì)胞系MC3/5FU由本實(shí)驗(yàn)室建系并保存。合成針對MRP1的shRNA，并篩選出被其穩(wěn)定轉(zhuǎn)染的MC3/5FU細(xì)胞系。通過逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)來檢測RNA的表達(dá)，蛋白質(zhì)印跡（Western blot）用來檢測蛋白表達(dá)的改變。甲基噻唑基四唑試驗(yàn)(MTT assay)用來檢測細(xì)胞生長和細(xì)胞耐藥性的改變。末端脫氧核苷酸轉(zhuǎn)移酶介導(dǎo)的dUTP缺口末端標(biāo)記測定法（TUNEL staining）用來檢測細(xì)胞凋亡。數(shù)據(jù)結(jié)果使用Student’s t-test and one-way ANOVA (LSD)來計(jì)算對比實(shí)驗(yàn)組與對照組之間的差異顯著性。計(jì)算由統(tǒng)計(jì)軟件SPSS version12.0完成。P0.05為有顯著差異。結(jié)果: MRP1的下調(diào)顯著提高了多藥耐藥細(xì)胞系MC3/5FU的對五氟尿嘧啶(5FU)，阿霉素(ADM)，表阿霉素(PADM)，博來霉素-A5(BLM-A5)，順鉑(CDDP)和紫杉醇(TAX)的化學(xué)敏感性，在一定5FU濃度下，MRP1的下調(diào)顯著抑制了MC3/5FU細(xì)胞的生長并顯著增加了其凋亡。 結(jié)論:MRP1在粘液表皮樣癌的多藥耐藥中發(fā)揮著重要作用。 第二部分. MRP1的核轉(zhuǎn)位參與粘液表皮樣癌的多藥耐藥性改變 目的：檢測MRP1的核轉(zhuǎn)移是否與粘液表皮樣癌的多藥耐藥性的改變相關(guān)。材料和方法:免疫熒光細(xì)胞化學(xué)染色法進(jìn)行細(xì)胞染色后，使用激光共聚焦顯微鏡用來檢測MRP1的亞細(xì)胞分布。免疫熒光組織化學(xué)法來檢測MRP1在粘液表皮樣癌腫瘤組織中和正常腮腺組織中的表達(dá)以及定位，并通過免疫化學(xué)組織染色法來對組織芯片染色，從而檢測MRP1在其他正常組織和其他腫瘤組織中的表達(dá)和定位。Student’st-test and one-way ANOVA (LSD)用來計(jì)算對比改變的顯著性以及實(shí)驗(yàn)組和對照組之間的差異。計(jì)算由統(tǒng)計(jì)軟件SPSS版本12.0完成，P0.05為有顯著差異。 結(jié)果:通過聚合酶鏈反應(yīng)和免疫印跡的方法，在RNA以及蛋白水平，對比MRP1在多藥耐藥細(xì)胞系MC3/5FU中和其親代細(xì)胞MC3中的表達(dá)，我們發(fā)現(xiàn)MRP1的表達(dá)并沒有發(fā)生量的變化。但是當(dāng)我們下調(diào)MRP1在MCA/5FU細(xì)胞中的表達(dá)后，細(xì)胞的多藥耐藥性卻明顯下降了。免疫熒光細(xì)胞化學(xué)染色進(jìn)行蛋白定位后發(fā)現(xiàn)MRP1從MC3細(xì)胞的細(xì)胞膜及細(xì)胞質(zhì)中轉(zhuǎn)移到了MC3/5FU細(xì)胞的細(xì)胞核中，而且MC3/5FU細(xì)胞中下調(diào)的MRP1主要為細(xì)胞核中的MRP1。為檢測核MRP1是否也出現(xiàn)在其他組織或者腫瘤中，也為了檢測是否為MRP1的一抗的非特異性染色才使得MRP1出現(xiàn)在細(xì)胞核中，我們用相同的一抗對組織芯片在相同條件下進(jìn)行免疫化學(xué)染色，結(jié)果我們并未發(fā)現(xiàn)MRP1在其他正常組織或者腫瘤組織中有核表達(dá)。 結(jié)論: MRP1的核轉(zhuǎn)移參與粘液表皮樣癌的多藥耐藥性改變。MRP1的核轉(zhuǎn)移可能是MRP1引發(fā)耐藥的新機(jī)制，基于這個(gè)新機(jī)制可能開發(fā)出新的粘液表皮樣癌治療方法。核MRP1可能為粘液表皮樣癌所特有，其有潛質(zhì)來成為鑒別診斷粘液表皮樣癌的一個(gè)Marker。 第三部分下調(diào)MRP1的表達(dá)導(dǎo)致MDR1表達(dá)的下降 目的:研究核MRP1是通過何種機(jī)制來參與粘液表皮樣癌的多藥耐藥的。 材料和方法:從第四軍醫(yī)大學(xué)口腔醫(yī)院病理科收集127例常規(guī)手術(shù)治療粘液表皮樣癌患者的腫瘤組織樣本,患者手術(shù)時(shí)間均在2006年至2011年。用免疫組織化學(xué)染色來檢測MRP1和MDR1在粘液表皮樣癌組織中的的表達(dá)。染色程度由蔡卜磊博士和劉園博士分別檢測。結(jié)果通過定性的方法結(jié)合定量的方法檢測。為排除MRP1表達(dá)在細(xì)胞核膜上的可能性，利用激光共聚焦顯微鏡進(jìn)行分層掃描進(jìn)行蛋白定位。 RT-PCR用來檢測RNA表達(dá)量的改變。Western blot用來檢測蛋白表達(dá)的改變。Student’s t-test andone-way ANOVA (LSD)用來計(jì)算對比改變的顯著性。斯皮爾曼等級相關(guān)分析來分析MDR1表達(dá)量和MRP1表達(dá)量的相關(guān)性。計(jì)算由統(tǒng)計(jì)軟件SPSS version12.0完成，P0.05為有顯著差異。 結(jié)果:為了排除MRP1一抗特異性的問題，我們從另一家公司購買了新的MRP1一抗來進(jìn)行免疫組織化學(xué)染色，結(jié)果我們再次確認(rèn)MRP1出現(xiàn)在粘液表皮樣癌的細(xì)胞核中。聚合酶鏈反應(yīng)以及免疫印跡法檢測發(fā)現(xiàn)，在下調(diào)MRP1的表達(dá)后，MDR1的表達(dá)也明顯降低。通過分析127例粘液表皮樣癌患者的腫瘤樣本相同位置的MRP1表達(dá)和MDR1表達(dá)及定位，再次確認(rèn)發(fā)現(xiàn)MRP1的表達(dá)量與MDR1的表達(dá)量有明顯正相關(guān)關(guān)系。 結(jié)論:在粘液表皮樣癌腫瘤中MRP1的表達(dá)量與MDR1的表達(dá)量顯著正相關(guān)。 第四部分核MRP1通過降低MDR1啟動(dòng)子的活性來調(diào)節(jié)MDR1的表達(dá) 目的:初步探索MRP1對MDR1表達(dá)通路中的影響 材料和方法: shRNA瞬時(shí)轉(zhuǎn)染MC3/5FU細(xì)胞系下調(diào)MRP1的表達(dá)。逆轉(zhuǎn)錄聚合酶鏈反應(yīng)用來檢測RNA的表達(dá)，免疫印跡法用來檢測蛋白表達(dá)的改變。熒光素酶報(bào)告分析（luciferase reporter assays）檢測MDR1啟動(dòng)子活性。Student’s t-test and one-wayANOVA (LSD)用來計(jì)算對比改變的顯著性。計(jì)算由統(tǒng)計(jì)軟件SPSS version12.0完成，P0.05為有顯著差異。 結(jié)果:瞬時(shí)轉(zhuǎn)染shRNA后，MRP1表達(dá)顯著下調(diào)，同時(shí)熒光素酶報(bào)告分析發(fā)現(xiàn)MDR1啟動(dòng)子活性顯著降低。 結(jié)論:核MRP1通過降低MDR1啟動(dòng)子的活性來調(diào)節(jié)MDR1的表達(dá)，其具體調(diào)控機(jī)制仍需我們進(jìn)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R739.87
本文編號：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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R739.87
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 溫玉明,代曉明,王昌美,李龍江,付風(fēng)華,王曉毅,唐休發(fā),劉華,華成舸,潘劍;口腔頜面部惡性腫瘤6539例臨床病理分析[J];華西口腔醫(yī)學(xué)雜志;2001年05期
本文編號：2249310
本文鏈接：http://sikaile.net/yixuelunwen/kouq/2249310.html
最近更新
教材專著
