【摘要】：研究背景：乳腺癌是女性最常見的惡性腫瘤之一,也是世界女性癌癥死亡的第二大原因。NAD(P)H:醌氧化還原酶1(NQO1)蛋白又稱醌氧化還原酶,不僅是一種黃素酶,也是一種Ⅱ相解毒酶,其發(fā)生缺陷后,與腫瘤發(fā)生的易感性密切相關(guān)。目前研究表明,NQO1蛋白在惡性腫瘤中呈高表達,并與患者的不良預后密切相關(guān)。β-拉帕醌是一種放射增敏劑,具有抗腫瘤的功效,可選擇性殺傷NQO1過表達的實體腫瘤。然而,它是否具有抑制乳腺癌轉(zhuǎn)移的作用、其機制如何?尚不明確。研究目的：探討NQO1蛋白在轉(zhuǎn)移性乳腺癌組織中過表達的臨床病理學意義,并通過體內(nèi)外分子生物學實驗探討β-拉帕醌對的乳腺癌細胞生物學行為的影響,分析其抑制乳腺癌細胞轉(zhuǎn)移的作用及分子機制,為乳腺癌的靶向治療提供可能的新靶點。材料和方法：應用免疫組化方法在45例癌旁正常組織、176例乳腺癌組織中檢測NQO1蛋白的表達情況,并應用qRT-PCR和Western-blot法對比分析62例新鮮乳腺癌組織和癌旁組織中NQO1的mRNA和蛋白表達情況,分析NQO1異常表達與轉(zhuǎn)移性乳腺癌之間的相關(guān)性。傳代培養(yǎng)NQO1過表達的MDA-MB-231、MCF-7乳腺癌細胞株；噻唑藍(MTT)及平板克隆實驗檢測β-拉帕醌對細胞增殖的抑制作用；通過劃痕實驗、遷移實驗、侵襲小室實驗檢測β-拉帕醌抑制MDA-MB-231、MCF-7細胞遷移和侵襲的能力；Western-blot法檢測β-拉帕醌對MDA-MB-231、MCF-7細胞NQO1蛋白和增殖、EMT標志物及Akt/mTOR信號通路相關(guān)蛋白表達的影響。體內(nèi)實驗部分：建立MDA-MB-231細胞系裸鼠移植瘤模型,隨機分成p-拉帕醌藥物處理組及對照組。連續(xù)給藥4周后檢測p-拉帕醌對MDA-MB-231移植瘤的抑制能力,并將瘤組織制成病理切片,應用免疫組化分析Ki67、E-cadherin、p-Akt、p-S6及p-4EBP1蛋白的表達情況。結(jié)果：1. qRT-PCR和Western blot法檢測顯示：62對乳腺癌新鮮組織中NQO1的mRNA和蛋白表達水平明顯高于癌旁正常組織；且轉(zhuǎn)移性乳腺癌組織中NQO1的mRNA和蛋白表達水平顯著高于非轉(zhuǎn)移性乳腺癌組織；轉(zhuǎn)移性乳腺癌細胞系中NQO1的mRNA和蛋白表達水平也顯著高于非轉(zhuǎn)移性乳腺癌細胞系；免疫組化結(jié)果亦表明,乳腺癌組織中NQO1蛋白表達陽性率和強陽性率(23%和62%)明顯高于癌旁正常組織(20%和0.0%)(P0.05)；2.MTT實驗與平板克隆實驗結(jié)果表明：4μM的β-拉帕醌給藥處理后MDA-MB-231、MCF-7細胞的增殖能力明顯下降(P0.05),Western blot結(jié)果顯示：β-拉帕醌通過下調(diào)Skp2和DEK蛋白的表達參與調(diào)節(jié)乳腺癌細胞的增殖。劃痕實驗、遷移實驗、侵襲小室實驗結(jié)果還顯示：4μM的β-拉帕醌可明顯抑制MDA-MB-231、MCF-7細胞的遷移和侵襲能力(P0.05),而且Western blot和免疫熒光染色結(jié)果表明,β-拉帕醌主要通過下調(diào)遷移相關(guān)蛋白MMP-9及上皮間質(zhì)轉(zhuǎn)化(EMT)間質(zhì)標記物Snail、Vimentin、Twist、Slug等的表達水平、上調(diào)EMT上皮標志物E-cadherin的表達影響MDA-MB-231、MCF-7細胞的遷移和侵襲能力；3.β-拉帕醌可下調(diào)MDA-MB-231、MCF-7細胞中Akt/mTOR相關(guān)信號通路蛋白p-AKT、p-S6和p-4EBP1的表達、上調(diào)p-PTEN的表達。4.β-拉帕醌抑制MDA-MB-231細胞的裸鼠移植瘤成瘤能力,且藥物處理組腫瘤組織壞死增多,Ki67、p-Akt、p-S6及p-4EBP1表達均下調(diào),而上皮標記物E-cadherin蛋白表達上調(diào)。結(jié)論：1.NQO1蛋白的表達水平與乳腺癌的轉(zhuǎn)移密切相關(guān)；2.β-拉帕醌可通過下調(diào)Skp2和DEK蛋白的表達抑制乳腺癌細胞的增殖；3.β-拉帕醌通過EMT途徑抑制乳腺癌細胞的侵襲和遷移能力；4.β-拉帕醌殺傷乳腺癌細胞可能是通過抑制Akt/mTOR通路實現(xiàn)的。
[Abstract]:Background: Breast cancer is one of the most common malignant tumors in women, and is the second cause of cancer death in the world. NAD (P) H: The oxidoreductase 1 (NQO1) protein, also known as the oxidoreductase, is not only a yellow enzyme, but also a II-phase detoxification enzyme, which is closely related to the susceptibility to tumorigenesis after the occurrence of a defect. The present study shows that the NQO1 protein is highly expressed in the malignant tumor and is closely related to the poor prognosis of the patient. The invention relates to a radiosensitizer, which has the functions of anti-tumor and can be used for selectively killing a solid tumor which is overexpressed by NQO1. However, does it have the role of inhibiting breast cancer metastasis, how is its mechanism? It's not clear. Objective: To study the clinical and pathological significance of the overexpression of NQO1 protein in the tissue of metastatic breast cancer, and to study the effect of the NQO1 protein on the biological behavior of breast cancer cells in the breast cancer, and to analyze the effect of the NQO1 protein on the metastasis of breast cancer cells and the molecular mechanism. And provides a possible new target for the targeted therapy of the breast cancer. Materials and Methods: The expression of NQO1 protein was detected by immunohistochemical method in 45 normal tissues and 176 breast cancer tissues, and the mRNA and protein expression of NQO1 in 62 fresh breast cancer tissues and adjacent tissues were analyzed by using qRT-PCR and Western-blot. The relationship between NQO1 abnormal expression and metastatic breast cancer was analyzed. MDA-MB-231 and MCF-7 breast cancer cell lines which were overexpressed by NQO1 were cultured, and the inhibition of the proliferation of cells was detected by MTT and plate cloning, and the inhibition of MDA-MB-231 was detected by scratch test, migration experiment and invasive cell test. The ability of cell migration and invasion of MCF-7 cells was detected by Western-blot. The effects of the expression of NQO1 and proliferation of MDA-MB-231, MCF-7 cells, NQO1 protein and proliferation, EMT marker and Akt/ mTOR signaling pathway were detected by Western-blot. In-vivo experimental part: The model of the nude mice with MDA-MB-231 cell line was established, and the model was randomly divided into p-Rapa drug treatment group and control group. The expression of Ki67, E-cadherin, p-Akt, p-S6 and p-4EBP1 was analyzed by immunohistochemistry. Results:1. The results of qRT-PCR and Western blot showed that the level of NQO1 mRNA and protein in the fresh tissue of breast cancer was significantly higher than that in the non-metastatic breast cancer tissues, and the mRNA and protein expression of NQO1 in the metastatic breast cancer tissues was significantly higher than that of the non-metastatic breast cancer tissues. The level of NQO1 mRNA and protein in the metastatic breast cancer cell line was also significantly higher than that of the non-metastatic breast cancer cell line, and the immunohistochemical results also showed that, The positive rate and positive rate of NQO1 protein in breast cancer tissues (23% and 62%) were significantly higher than that in the normal tissues (20% and 0.0%) (P0.05). The proliferation ability of MCF-7 cells decreased significantly (P0.05). Western blot showed that the expression of Skp2 and DEK protein was involved in the regulation of the proliferation of breast cancer cells by down-regulating the expression of Skp2 and DEK protein. The results of the scratch test, the migration experiment and the invasion cell also showed that 4. m u.M of L-Rapa could significantly inhibit the migration and invasion ability of MDA-MB-231 and MCF-7 cells (P0.05), and the results of Western blot and immunofluorescence staining showed that, The expression of E-cadherin in EMT epithelial marker was up-regulated by down-regulation of the expression levels of MMP-9 and epithelial-mesenchymal transition (EMT) interstitial markers Snail, Vimentin, Twist, Slug, etc., and the expression of E-cadherin in the EMT epithelial marker was up-regulated in MDA-MB-231 and MCF-7 cells. The expression of p-AKT, p-S6 and p-4EBP1 in the expression of p-AKT, p-S6 and p-4EBP1 in MDA-MB-231 and MCF-7 cells could be down-regulated by P-Lopterin, and the expression of p-PTEN was up-regulated. The expression of E-cadherin was down-regulated, and the expression of E-cadherin was up-regulated. Conclusion:1. The expression level of NQO1 protein is closely related to the metastasis of breast cancer. The expression of Skp2 and DEK can inhibit the proliferation of breast cancer cells by down-regulating the expression of Skp2 and DEK proteins. The resistance to invasion and migration of breast cancer cells is inhibited by the method of EMT. The anti-human breast cancer cells of the P-Rapa may be achieved by inhibiting the Akt/ mTOR pathway.
【學位授予單位】：延邊大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.9

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