乳腺癌中P53的表达及其与miR-449a的调控机制研究初探

发布时间：2018-05-11 06:31

本文选题：乳腺癌 + 三阴性乳腺癌　；参考：《川北医学院》2017年硕士论文

【摘要】：研究背景:乳腺癌在女性恶性肿瘤中发病率和致死人数居首位,严重威胁着女性生命健康,尤其是占乳腺癌总数约10%~20%的三阴性乳腺癌,由于恶性程度高且缺乏有效地治疗方法,预后极差。如何能够早期诊断,并准确分型和针对性靶向治疗成为目前攻克乳腺癌的关键,也是解决所有癌症问题的共通出路。肿瘤的发生发展是多基因参与、多步骤协同作用的过程,癌基因的激活、抑癌基因的失活在这过程中有重要意义。P53、PTEN和c-Myc是与乳腺癌发生和进展密切关联的癌基因与抑癌基因,尤其是在三阴乳腺癌中,P53突变频率极高,约80%的三阴性乳腺癌存在P53的突变,生物学行为更为恶劣,治疗更为棘手。然而,对于P53在乳腺癌外周血中的表达情况不太清楚。相对于肿瘤组织,外周血白细胞是极易获得的标本,且有研究证实肿瘤患者外周血中基因的表达明显异于健康人群。因此,本课题首先检测P53、PTEN、c-Myc的m RNA在乳腺癌患者外周血白细胞中的表达水平,探讨其与临床病理资料间的相互关系及其潜在的临床应用价值,以期为乳腺癌的早期、无创检查探索潜在的有效途径。微小RNAs(micro RNAs,miRNAs)是一种长约22个核苷酸的非编码RNAs,通过结合目的基因的m RNA,阻止翻译或降解m RNA,对目的基因进行转录后负性调控。研究证实miRNAs在肿瘤的发生和进展过程中发挥重要的调控作用,扮演着癌基因和抑癌基因的角色,探讨miRNAs在肿瘤中发挥的作用及其作用机制对于探索潜在的肿瘤标志物和治疗靶点都有重要的价值。最近的研究显示miR-449a在表达突变型P53蛋白的以及基底样型的乳腺癌组织中明显降低,而基底样型乳腺癌绝大部分是恶性程度最高的三阴性乳腺癌,且约80%的三阴性乳腺癌存在P53的突变。在肺癌、肝癌、前列腺癌等多种癌症中的研究显示miR-449a抑制癌细胞侵袭迁移和增殖,发挥抑癌作用。然而,miR-449a在三阴乳腺癌中作用如何,其与突变型P53之间是否具有潜在的相互调节作用,机制如何,能否为三阴乳腺癌的诊治提供新的思路?目前尚未见报道。因此,本课题第二部分探讨了miR-449a在三阴乳腺癌中所起的作用,及其及其突变型P53间的调控机制,旨在探索寻求潜在的生物标志物和治疗新靶点。目的:1.探讨肿瘤相关基因P53、PTEN、c-Myc在乳腺癌外周血白细胞中的表达情况与临床病理资料间的相互关系及其潜在的临床应用价值;2.明确miR-449a对三阴性乳腺癌细胞中突变型P53的调控作用,并初步探讨其作用机制。方法:1.Real-time PCR检测乳腺癌患者P53、PTEN、c-Myc基因的在乳腺癌、良性乳腺疾病女性患者和健康体检对照女性外周血白细胞中的m RNA表达水平;2.MDA-MB-468细胞转染P53敲低质粒p Super-Retro-puro-sh-P53(以下简称为sh-P53)和空载体质粒p Super-Retro-puro-Vector(以下简称为Vector),Real-time PCR检测P53、E-cadherin、Vimentin、Twist、Snail的m RNA和miR-449a水平,Western Blot检测P53、AKT1、p-AKT、p-m TOR、Bcl-2、caspase-3、Cleaved-caspase-3的蛋白水平;3.MDA-MB-468细胞转染miR-449a-mimic和miR-449a-NC,转染48h后Real-time PCR检测P53、E-cadherin、Vimentin、Twist、Snail的m RNA和miR-449a水平,转染72后Western Blot检测P53、AKT1、p-AKT、p-m TOR、Bcl-2.、caspase-3、Cleaved-caspase-3的蛋白水平;4.结晶紫染色法绘制生长曲线检测转染处理后MDA-MB-468细胞的增殖能力;5.划痕试验和Transwell实验检测转染处理后MDA-MB-468细胞的迁移能力。结果:1.乳腺癌和良性乳腺疾病患者外周血白细胞中P53和PTEN的m RNA表达水平明显降低;2.PTEN的m RNA水平下调与乳腺癌淋巴结转移及其病理分期密切关联;3.MDA-MB-468细胞中降低P53表达不影响miR-449a表达水平;4.降低P53表达明显抑制MDA-MB-468细胞增殖能力和迁移能力;5.敲低MDA-MB-468中突变型P53后,细胞信号通路PI3K/AKT/m TOR和EMT被抑制,凋亡抑制蛋白Bcl-2表达水平下调,激活了凋亡执行蛋白caspase-3;6.升高MDA-MB-468细胞内miR-449a水平抑制了细胞增殖和迁移能力;7.升高miR-449a水平下调了MDA-MB-468细胞内突变型P53水平,同时抑制了PI3K/AKT/m TOR和EMT,下调了Bcl-2水平。结论:1.乳腺癌患者外周血白细胞中P53等肿瘤相关基因的m RNA表达水平与健康人群和良性乳腺疾病患者相比存在差异,且与临床病理资料高度相关,提示乳腺癌外周血白细胞中P53等肿瘤相关基因检测可为监测肿瘤发生、进展和预后判断提供极具潜力的无创检测手段;2.降低突变型P53的水平可抑制三阴乳腺癌MDA-MB-468的细胞增殖和迁移能力,降低其恶性程度,但不影响miR-449a的表达;3.降低突变型P53的水平可抑制PI3K/AKT/m TOR细胞信号转导通路和EMT,诱导细胞凋亡;4.miR-449a可通过下调突变型P53的表达水平,抑制PI3K/AKT/m TOR细胞信号转导通路和EMT,诱导细胞凋亡,从而抑制MDA-MB-468细胞的细胞增殖和迁移能力;5.miR-449a通过抑制突变型P53及其相关细胞信号通路,在三阴乳腺癌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.

【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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