本文選題：miR-34a + Notch通路��； 參考：《吉林大學》2015年博士論文

【摘要】：乳腺癌是女性易患腫瘤之一。雖然近年來在乳腺癌早期診斷與系統(tǒng)治療方面有較大進展，但其復發(fā)率及難治率仍然居高不下。因而，尋找新的有效的診斷及治療方法一直是乳腺癌研究中的熱點問題。乳腺癌干細胞是乳腺癌中一小部分具有自我更新、無限增殖、多向分化等干細胞特性的癌細胞亞群。大量研究顯示它是乳腺癌發(fā)生的根源，在乳腺癌的復發(fā)、轉移及對化療藥物耐藥性的產(chǎn)生等方面都扮演了重要角色。 MicroRNAs（miRNAs）是一類內源性非編碼單鏈小分子RNA，一般長約18～24個核苷酸。成熟的miRNA通過特異性結合靶mRNA的3’端非翻譯區(qū)（3’-untranslated region，3’-UTR），使靶蛋白mRNA降解或使其翻譯受到抑制，從而調節(jié)內源基因的表達。miRNA參與了細胞增殖、遷移、凋亡、分化等多種細胞生命活動的調節(jié)。大量研究證實miRNA在幾乎所有的惡性腫瘤中都存在表達異常，認為其在腫瘤發(fā)生發(fā)展中起到重要作用。近來研究認為其在腫瘤干細胞的自我更新、分化等過程中也扮演重要角色。根據(jù)miRNA在腫瘤細胞中的作用可將其分為兩類，，一類在腫瘤細胞中表達升高，并且可通過抑制抑癌基因的表達促進腫瘤的發(fā)生，被稱為促癌miRNA；另一類則其可通過抑制癌基因的表達抑制腫瘤的發(fā)生發(fā)展，被稱為抑癌miRNA。作為研究較早的抑癌miRNA，miR-34a在多種腫瘤中發(fā)揮抑癌作用，近年來研究發(fā)現(xiàn)其在前列腺癌干細胞、胰腺癌干細胞中也發(fā)揮重要的調節(jié)作用，但其在乳腺癌干細胞中的作用及機制尚需進一步研究。 Notch信號通路是一個進化上相對保守的通路，在胚胎發(fā)育、器官成熟及腫瘤發(fā)生發(fā)展過程中均發(fā)揮重要的調控作用。Notch1是與乳腺癌的發(fā)生、轉移及耐藥密切相關的Notch受體之一。近年來研究發(fā)現(xiàn)其在維持乳腺癌干細胞的自我更新，調控其分化中扮演重要角色。Notch1是miR-34a的靶基因之一，因而miR-34a/Notch1可能在乳腺癌細胞及乳腺癌干細胞的調控中發(fā)揮重要作用。 本研究通過對臨床組織樣本的檢測及體外實驗，探討了miR-34a靶向Notch1在乳腺癌細胞的增殖、侵襲遷移、乳腺癌干細胞調控及腫瘤耐藥性中的作用，為乳腺癌的臨床診斷及治療提供了依據(jù)。 方法： 以qRT-PCR方法檢測乳腺癌組織及正常組織中miR-34a及Notch1mRNA的表達水平，以免疫組化方法檢測Notch1在乳腺癌中的表達水平，分析miR-34a與腫瘤轉移、臨床分期以及Notch1表達的相關關系。 以miR-34a模擬物、Notch1siRNA或其陰性對照分別轉染MCF-7細胞，采用qRT-PCR及Western blot方法檢測Notch1及其下游因子的表達變化，采用CCK-8法檢測細胞增殖水平，采用Transwell法檢測細胞侵襲及遷移的變化，Western blot法檢測E-cadherin、Vimentin的表達變化。采用流式細胞術檢測CD44+/CD24-細胞比例，Western blot法檢測ALDH1表達水平變化。 構建表達Notch1胞內結構域的pcDNA3.1-Notch1質粒，并將其與miR-34a模擬物共轉染于MCF-7細胞中，檢測各組細胞增殖水平、遷移能力、CD44+/CD24-細胞比例及ALDH1的表達變化，以觀察miR-34a/Notch1在乳腺癌細胞增殖、侵襲遷移以及乳腺癌干細胞調控中的作用。 將miR-34a模擬物與紫杉醇分別或聯(lián)合作用于MCF-7細胞，以CCK-8法觀察對細胞活力的影響，以乳腺球形成實驗檢測乳腺癌干細胞自我更新能力，以Western blot方法檢測Notch1、Hes1、ABCG2及ALDH1的表達水平，探討miR-34a影響MCF-7細胞化療敏感性的作用機制。 結果： miR-34a在乳腺癌組織中的表達水平明顯低于正常組織，在伴有淋巴結轉移的乳腺癌組織中明顯低于不伴淋巴結轉移的乳腺癌組織。在臨床Ⅰ-Ⅲ期患者中，乳腺癌組織中miR-34a表達水平隨腫瘤分期的升高而降低。在乳腺癌組織中，miR-34a與Notch1的表達水平呈負相關。轉染miR-34a模擬物上調miR-34a后可觀察到Notch通路中Notch1及Hes1的表達水平明顯下降。雙熒光素酶報告基因分析顯示miR-34a與Notch13’-UTR間存在直接作用的靶點。上調miR-34a能夠明顯抑制MCF-7細胞的增殖、侵襲及遷移水平，與下調Notch1的作用相似。而在過表達miR-34a的細胞中上調Notch1能夠部分逆轉miR-34a對乳腺癌細胞的抑制作用。提示miR-34a/Notch1在調節(jié)乳腺癌細胞增殖、侵襲及遷移過程中占有重要地位。對乳腺癌干細胞標志物ALDH1、CD44、CD24的檢測發(fā)現(xiàn)，上調miR-34a及下調Notch1均能夠明顯抑制ALDH1的表達水平，降低CD44+/CD24-細胞比例，而在過表達miR-34a的細胞中同時上調Notch1則能夠部分逆轉miR-34a對乳腺癌干細胞的抑制作用。提示Notch1通路是介導miR-34a抑制乳腺癌干細胞作用的重要機制之一。將紫杉醇與miR-34a模擬物共同作用于MCF-7細胞后發(fā)現(xiàn)，miR-34a能夠明顯增加紫杉醇對細胞的抑制作用。與應用紫杉醇治療相比，miR-34a模擬物及紫杉醇的聯(lián)合應用能使乳腺球形成效率及Notch1、Hes1、ABCG2、ALDH1的表達水平均明顯降低。提示miR-34增加紫杉醇藥物敏感性的機制與其抑制Notch1通路及抑制乳腺癌自我更新能力有關。 結論： miR-34a能夠通過靶向Notch1抑制乳腺癌細胞增殖、侵襲及遷移能力并抑制乳腺癌干細胞特性。同時miR-34a能夠增加紫杉醇藥物敏感性，其機制與抑制Notch1通路及抑制乳腺癌干細胞的自我更新能力有關。這些結果提示上調miR-34a的表達是增強乳腺癌療效的有效途徑。
[Abstract]:Breast cancer is one of the most vulnerable tumors in women. Although recent progress has been made in the early diagnosis and system treatment of breast cancer, the recurrence rate and refractory rate are still high. Therefore, finding new effective diagnosis and treatment methods has always been a hot issue in the research of breast cancer. A large number of studies have shown that it is the source of breast cancer, and plays an important role in the recurrence and metastasis of breast cancer and the production of drug resistance to chemotherapeutic drugs.
MicroRNAs (miRNAs) is a class of endogenous non coding single strand small molecule RNA, which generally has about 18~24 nucleotides. Mature miRNA can degrade or suppress the target protein mRNA by specific binding to the target mRNA's 3 'terminal non translation region (3' -untranslated region, 3 '-UTR), thus regulating the expression of the endogenous gene and participates in the cells. Proliferation, migration, apoptosis, differentiation and a variety of cell life regulation. A large number of studies have shown that miRNA has an abnormal expression in almost all malignant tumors and is considered to play an important role in the development of tumor. Recently, it is considered to play an important role in the process of self new and differentiation of tumor stem cells. According to miRN The role of A in tumor cells can be divided into two categories. One class is expressed in tumor cells and can promote the occurrence of tumor by inhibiting the expression of tumor suppressor genes. It is called cancer promoting miRNA. The other one can inhibit the development of tumor by inhibiting the expression of oncogene. It is called anti cancer miRNA. as the early research of tumor suppressor m. IRNA, miR-34a plays a role in tumor suppressor in a variety of tumors. In recent years, it has been found that it plays an important role in prostate cancer stem cells and pancreatic cancer stem cells, but its role and mechanism in breast cancer stem cells still need further study.
Notch signaling pathway is an evolutionary relatively conservative pathway that plays an important regulatory role in embryonic development, organ maturation and tumor development..Notch1 is one of the Notch receptors closely related to the occurrence, metastasis and drug resistance of breast cancer. In recent years, it has been found to maintain self-renewal and control of breast cancer stem cells. The role of.Notch1 is one of the target genes of miR-34a, so miR-34a/Notch1 may play an important role in the regulation of breast cancer cells and breast cancer stem cells.
In this study, the effects of miR-34a targeting Notch1 on the proliferation, invasion and migration of breast cancer cells, the regulation of breast cancer stem cells and the drug resistance of the breast cancer cells were explored through the detection of clinical tissue samples and in vitro experiments, which provided a basis for the clinical diagnosis and treatment of breast cancer.
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本文編號：2012205