本文選題：乳腺癌 + 三陰性乳腺癌�。� 參考：《川北醫(yī)學(xué)院》2017年碩士論文

【摘要】：研究背景:乳腺癌在女性惡性腫瘤中發(fā)病率和致死人數(shù)居首位,嚴重威脅著女性生命健康,尤其是占乳腺癌總數(shù)約10%~20%的三陰性乳腺癌,由于惡性程度高且缺乏有效地治療方法,預(yù)后極差。如何能夠早期診斷,并準(zhǔn)確分型和針對性靶向治療成為目前攻克乳腺癌的關(guān)鍵,也是解決所有癌癥問題的共通出路。腫瘤的發(fā)生發(fā)展是多基因參與、多步驟協(xié)同作用的過程,癌基因的激活、抑癌基因的失活在這過程中有重要意義。P53、PTEN和c-Myc是與乳腺癌發(fā)生和進展密切關(guān)聯(lián)的癌基因與抑癌基因,尤其是在三陰乳腺癌中,P53突變頻率極高,約80%的三陰性乳腺癌存在P53的突變,生物學(xué)行為更為惡劣,治療更為棘手。然而,對于P53在乳腺癌外周血中的表達情況不太清楚。相對于腫瘤組織,外周血白細胞是極易獲得的標(biāo)本,且有研究證實腫瘤患者外周血中基因的表達明顯異于健康人群。因此,本課題首先檢測P53、PTEN、c-Myc的m RNA在乳腺癌患者外周血白細胞中的表達水平,探討其與臨床病理資料間的相互關(guān)系及其潛在的臨床應(yīng)用價值,以期為乳腺癌的早期、無創(chuàng)檢查探索潛在的有效途徑。微小RNAs(micro RNAs,miRNAs)是一種長約22個核苷酸的非編碼RNAs,通過結(jié)合目的基因的m RNA,阻止翻譯或降解m RNA,對目的基因進行轉(zhuǎn)錄后負性調(diào)控。研究證實miRNAs在腫瘤的發(fā)生和進展過程中發(fā)揮重要的調(diào)控作用,扮演著癌基因和抑癌基因的角色,探討miRNAs在腫瘤中發(fā)揮的作用及其作用機制對于探索潛在的腫瘤標(biāo)志物和治療靶點都有重要的價值。最近的研究顯示miR-449a在表達突變型P53蛋白的以及基底樣型的乳腺癌組織中明顯降低,而基底樣型乳腺癌絕大部分是惡性程度最高的三陰性乳腺癌,且約80%的三陰性乳腺癌存在P53的突變。在肺癌、肝癌、前列腺癌等多種癌癥中的研究顯示miR-449a抑制癌細胞侵襲遷移和增殖,發(fā)揮抑癌作用。然而,miR-449a在三陰乳腺癌中作用如何,其與突變型P53之間是否具有潛在的相互調(diào)節(jié)作用,機制如何,能否為三陰乳腺癌的診治提供新的思路?目前尚未見報道。因此,本課題第二部分探討了miR-449a在三陰乳腺癌中所起的作用,及其及其突變型P53間的調(diào)控機制,旨在探索尋求潛在的生物標(biāo)志物和治療新靶點。目的:1.探討腫瘤相關(guān)基因P53、PTEN、c-Myc在乳腺癌外周血白細胞中的表達情況與臨床病理資料間的相互關(guān)系及其潛在的臨床應(yīng)用價值;2.明確miR-449a對三陰性乳腺癌細胞中突變型P53的調(diào)控作用,并初步探討其作用機制。方法:1.Real-time PCR檢測乳腺癌患者P53、PTEN、c-Myc基因的在乳腺癌、良性乳腺疾病女性患者和健康體檢對照女性外周血白細胞中的m RNA表達水平;2.MDA-MB-468細胞轉(zhuǎn)染P53敲低質(zhì)粒p Super-Retro-puro-sh-P53(以下簡稱為sh-P53)和空載體質(zhì)粒p Super-Retro-puro-Vector(以下簡稱為Vector),Real-time PCR檢測P53、E-cadherin、Vimentin、Twist、Snail的m RNA和miR-449a水平,Western Blot檢測P53、AKT1、p-AKT、p-m TOR、Bcl-2、caspase-3、Cleaved-caspase-3的蛋白水平;3.MDA-MB-468細胞轉(zhuǎn)染miR-449a-mimic和miR-449a-NC,轉(zhuǎn)染48h后Real-time PCR檢測P53、E-cadherin、Vimentin、Twist、Snail的m RNA和miR-449a水平,轉(zhuǎn)染72后Western Blot檢測P53、AKT1、p-AKT、p-m TOR、Bcl-2.、caspase-3、Cleaved-caspase-3的蛋白水平;4.結(jié)晶紫染色法繪制生長曲線檢測轉(zhuǎn)染處理后MDA-MB-468細胞的增殖能力;5.劃痕試驗和Transwell實驗檢測轉(zhuǎn)染處理后MDA-MB-468細胞的遷移能力。結(jié)果:1.乳腺癌和良性乳腺疾病患者外周血白細胞中P53和PTEN的m RNA表達水平明顯降低;2.PTEN的m RNA水平下調(diào)與乳腺癌淋巴結(jié)轉(zhuǎn)移及其病理分期密切關(guān)聯(lián);3.MDA-MB-468細胞中降低P53表達不影響miR-449a表達水平;4.降低P53表達明顯抑制MDA-MB-468細胞增殖能力和遷移能力;5.敲低MDA-MB-468中突變型P53后,細胞信號通路PI3K/AKT/m TOR和EMT被抑制,凋亡抑制蛋白Bcl-2表達水平下調(diào),激活了凋亡執(zhí)行蛋白caspase-3;6.升高MDA-MB-468細胞內(nèi)miR-449a水平抑制了細胞增殖和遷移能力;7.升高miR-449a水平下調(diào)了MDA-MB-468細胞內(nèi)突變型P53水平,同時抑制了PI3K/AKT/m TOR和EMT,下調(diào)了Bcl-2水平。結(jié)論:1.乳腺癌患者外周血白細胞中P53等腫瘤相關(guān)基因的m RNA表達水平與健康人群和良性乳腺疾病患者相比存在差異,且與臨床病理資料高度相關(guān),提示乳腺癌外周血白細胞中P53等腫瘤相關(guān)基因檢測可為監(jiān)測腫瘤發(fā)生、進展和預(yù)后判斷提供極具潛力的無創(chuàng)檢測手段;2.降低突變型P53的水平可抑制三陰乳腺癌MDA-MB-468的細胞增殖和遷移能力,降低其惡性程度,但不影響miR-449a的表達;3.降低突變型P53的水平可抑制PI3K/AKT/m TOR細胞信號轉(zhuǎn)導(dǎo)通路和EMT,誘導(dǎo)細胞凋亡;4.miR-449a可通過下調(diào)突變型P53的表達水平,抑制PI3K/AKT/m TOR細胞信號轉(zhuǎn)導(dǎo)通路和EMT,誘導(dǎo)細胞凋亡,從而抑制MDA-MB-468細胞的細胞增殖和遷移能力;5.miR-449a通過抑制突變型P53及其相關(guān)細胞信號通路,在三陰乳腺癌MDA-MB-468中起到抑癌基因的作用。
[Abstract]:Background: the incidence and death toll of breast cancer is the first in female malignant tumor, which seriously threatens the life and health of women, especially the three negative breast cancer, which accounts for about 10%~20% of the total number of breast cancer. The prognosis is very poor because of the high degree of malignancy and the lack of effective treatment. The development of cancer is a common way to solve all cancer problems. The development of cancer is a multi gene participation, the process of multi step synergy, the activation of the oncogene and the inactivation of the tumor suppressor gene is of great significance in this process.P53, PTEN and c-Myc are the oncogenes closely associated with the development and progression of breast cancer. With tumor suppressor genes, especially in three negative breast cancer, P53 mutation frequency is very high, about 80% of three negative breast cancers have P53 mutation, the biological behavior is worse, and the treatment is more difficult. However, the expression of P53 in the peripheral blood of breast cancer is not very clear. Studies have confirmed that the expression of gene in peripheral blood of tumor patients is obviously different from that of healthy people. Therefore, we first detect the expression level of P53, PTEN and c-Myc m RNA in peripheral blood leukocytes of breast cancer patients, and discuss the relationship between them and the clinicopathological data and the potential clinical application value, so as to be the early stage of breast cancer. RNAs (micro RNAs (miRNAs) is a non coding RNAs with 22 nucleotides, which combines the m RNA of the target gene to prevent the translation or degradation of M RNA and the post transcriptional negative regulation of the target gene. The study confirms that miRNAs plays an important regulatory role in the development and progression of the tumor, The role of the oncogene and tumor suppressor gene to explore the role of miRNAs in the tumor and its mechanism is of great value in exploring potential tumor markers and targets. Recent studies have shown that miR-449a is significantly reduced in the expression of mutant P53 protein and in basal like breast cancer, and the basal like type. Most of the breast cancer is the most malignant three negative breast cancer, and about 80% of the three negative breast cancers have P53 mutation. The research in many kinds of cancers, such as lung cancer, liver cancer and prostate cancer, shows that miR-449a inhibits the invasion and migration and proliferation of cancer cells and plays the role of inhibiting cancer. However, the role of miR-449a in three yin breast cancer Is there a potential mutual regulation between the variant P53 and how the mechanism can provide a new way of thinking for the diagnosis and treatment of three negative breast cancer? The second part of this topic has discussed the role of miR-449a in three negative breast cancer and its regulatory mechanism between the mutant P53 and the aim of exploring the potential survival. Objective: 1. to investigate the relationship between the expression of tumor related genes P53, PTEN, c-Myc in the peripheral blood leukocytes of breast cancer and the relationship between the clinicopathological data and the potential clinical application value; 2. to clarify the regulation of miR-449a on the mutant P53 in three negative breast cancer cells and to discuss its effect preliminarily. Methods: 1.Real-time PCR was used to detect the expression level of M RNA in breast cancer, PTEN, c-Myc gene in breast cancer, benign breast disease female patients and healthy physical examination compared with female peripheral blood white blood cells; 2.MDA-MB-468 cells transfected P53 knockout plasmid P Super-Retro-puro-sh-P53 (hereinafter referred to as sh-P53) and empty body constitution granules. Etro-puro-Vector (hereinafter referred to as Vector), Real-time PCR detection P53, E-cadherin, Vimentin, Twist, Snail m RNA and miR-449a levels. M RNA and miR-449a levels of -cadherin, Vimentin, Twist, Snail, and Western Blot detection P53, AKT1, p-AKT, Snail, after transfection of 72; the growth curve was drawn to detect the proliferation ability of the cells after transfection; 5. scratch test and experimental detection of transfection treatment The migration ability of post MDA-MB-468 cells. Results: 1. the m RNA expression level of P53 and PTEN in peripheral blood leukocytes of patients with breast cancer and benign breast disease decreased significantly; the downregulation of M RNA level of 2.PTEN was closely related to lymph node metastasis and pathological stage of breast cancer; the decrease of P53 expression in 3.MDA-MB-468 cells did not affect miR-449a expression level; 4. decreased. Low P53 expression significantly inhibited the proliferation and migration of MDA-MB-468 cells. 5. after knocking down the mutant P53, the cell signaling pathway PI3K/AKT/m TOR and EMT were inhibited, the expression level of apoptosis inhibitory protein Bcl-2 was down, and apoptotic execution protein caspase-3 was activated; 6. increased MDA-MB-468 cell miR-449a levels inhibited cell proliferation and inhibition. 7. elevated miR-449a level lowered the P53 level in MDA-MB-468 cells, inhibited PI3K/AKT/m TOR and EMT and lowered the Bcl-2 level. Conclusion: the level of M RNA expression of P53 and other tumor related genes in peripheral blood leukocytes of 1. breast cancer patients is different from those in healthy people and patients with benign breast disease. The pathological data of the bed are highly correlated, suggesting that the detection of P53 and other tumor related genes in the peripheral blood leukocytes of the breast cancer can provide a highly potential noninvasive detection method for monitoring the occurrence, progression and prognosis of the tumor, and the level of 2. reduced Mutant P53 can inhibit the proliferation and migration of MDA-MB-468 in three negative breast cancer, and reduce the degree of malignancy, but it can reduce the malignancy of the breast cancer. It does not affect the expression of miR-449a; 3. reducing the level of mutant P53 can inhibit the PI3K/AKT/m TOR cell signal transduction pathway and EMT to induce apoptosis, and 4.miR-449a can inhibit the proliferation of MDA-MB-468 cells by down regulating the expression level of the mutant P53, inhibiting the PI3K/AKT/m TOR cell signal transduction pathway and EMT, inducing cell apoptosis. 5.miR-449a inhibits oncogene expression in the three negative breast cancer MDA-MB-468 by inhibiting mutant P53 and its related cellular signaling pathways.

【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.9

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