β-拉帕醌通过EMT抑制乳腺癌侵袭转移的机制研究

发布时间：2019-05-17 12:00

【摘要】：研究背景：乳腺癌是女性最常见的恶性肿瘤之一,也是世界女性癌症死亡的第二大原因。NAD(P)H:醌氧化还原酶1(NQO1)蛋白又称醌氧化还原酶,不仅是一种黄素酶,也是一种Ⅱ相解毒酶,其发生缺陷后,与肿瘤发生的易感性密切相关。目前研究表明,NQO1蛋白在恶性肿瘤中呈高表达,并与患者的不良预后密切相关。β-拉帕醌是一种放射增敏剂,具有抗肿瘤的功效,可选择性杀伤NQO1过表达的实体肿瘤。然而,它是否具有抑制乳腺癌转移的作用、其机制如何?尚不明确。研究目的：探讨NQO1蛋白在转移性乳腺癌组织中过表达的临床病理学意义,并通过体内外分子生物学实验探讨β-拉帕醌对的乳腺癌细胞生物学行为的影响,分析其抑制乳腺癌细胞转移的作用及分子机制,为乳腺癌的靶向治疗提供可能的新靶点。材料和方法：应用免疫组化方法在45例癌旁正常组织、176例乳腺癌组织中检测NQO1蛋白的表达情况,并应用qRT-PCR和Western-blot法对比分析62例新鲜乳腺癌组织和癌旁组织中NQO1的mRNA和蛋白表达情况,分析NQO1异常表达与转移性乳腺癌之间的相关性。传代培养NQO1过表达的MDA-MB-231、MCF-7乳腺癌细胞株；噻唑蓝(MTT)及平板克隆实验检测β-拉帕醌对细胞增殖的抑制作用；通过划痕实验、迁移实验、侵袭小室实验检测β-拉帕醌抑制MDA-MB-231、MCF-7细胞迁移和侵袭的能力；Western-blot法检测β-拉帕醌对MDA-MB-231、MCF-7细胞NQO1蛋白和增殖、EMT标志物及Akt/mTOR信号通路相关蛋白表达的影响。体内实验部分：建立MDA-MB-231细胞系裸鼠移植瘤模型,随机分成p-拉帕醌药物处理组及对照组。连续给药4周后检测p-拉帕醌对MDA-MB-231移植瘤的抑制能力,并将瘤组织制成病理切片,应用免疫组化分析Ki67、E-cadherin、p-Akt、p-S6及p-4EBP1蛋白的表达情况。结果：1. qRT-PCR和Western blot法检测显示：62对乳腺癌新鲜组织中NQO1的mRNA和蛋白表达水平明显高于癌旁正常组织；且转移性乳腺癌组织中NQO1的mRNA和蛋白表达水平显著高于非转移性乳腺癌组织；转移性乳腺癌细胞系中NQO1的mRNA和蛋白表达水平也显著高于非转移性乳腺癌细胞系；免疫组化结果亦表明,乳腺癌组织中NQO1蛋白表达阳性率和强阳性率(23%和62%)明显高于癌旁正常组织(20%和0.0%)(P0.05)；2.MTT实验与平板克隆实验结果表明：4μM的β-拉帕醌给药处理后MDA-MB-231、MCF-7细胞的增殖能力明显下降(P0.05),Western blot结果显示：β-拉帕醌通过下调Skp2和DEK蛋白的表达参与调节乳腺癌细胞的增殖。划痕实验、迁移实验、侵袭小室实验结果还显示：4μM的β-拉帕醌可明显抑制MDA-MB-231、MCF-7细胞的迁移和侵袭能力(P0.05),而且Western blot和免疫荧光染色结果表明,β-拉帕醌主要通过下调迁移相关蛋白MMP-9及上皮间质转化(EMT)间质标记物Snail、Vimentin、Twist、Slug等的表达水平、上调EMT上皮标志物E-cadherin的表达影响MDA-MB-231、MCF-7细胞的迁移和侵袭能力；3.β-拉帕醌可下调MDA-MB-231、MCF-7细胞中Akt/mTOR相关信号通路蛋白p-AKT、p-S6和p-4EBP1的表达、上调p-PTEN的表达。4.β-拉帕醌抑制MDA-MB-231细胞的裸鼠移植瘤成瘤能力,且药物处理组肿瘤组织坏死增多,Ki67、p-Akt、p-S6及p-4EBP1表达均下调,而上皮标记物E-cadherin蛋白表达上调。结论：1.NQO1蛋白的表达水平与乳腺癌的转移密切相关；2.β-拉帕醌可通过下调Skp2和DEK蛋白的表达抑制乳腺癌细胞的增殖；3.β-拉帕醌通过EMT途径抑制乳腺癌细胞的侵袭和迁移能力；4.β-拉帕醌杀伤乳腺癌细胞可能是通过抑制Akt/mTOR通路实现的。
[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.
【学位授予单位】：延边大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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