MiR-34a靶向调控Notch1抑制乳腺癌及增加其化疗敏感性的研究

发布时间：2018-06-13 02:16

  本文选题：miR-34a + Notch通路　； 参考：《吉林大学》2015年博士论文

【摘要】：乳腺癌是女性易患肿瘤之一。虽然近年来在乳腺癌早期诊断与系统治疗方面有较大进展，但其复发率及难治率仍然居高不下。因而，寻找新的有效的诊断及治疗方法一直是乳腺癌研究中的热点问题。乳腺癌干细胞是乳腺癌中一小部分具有自我更新、无限增殖、多向分化等干细胞特性的癌细胞亚群。大量研究显示它是乳腺癌发生的根源，在乳腺癌的复发、转移及对化疗药物耐药性的产生等方面都扮演了重要角色。 MicroRNAs（miRNAs）是一类内源性非编码单链小分子RNA，一般长约18～24个核苷酸。成熟的miRNA通过特异性结合靶mRNA的3’端非翻译区（3’-untranslated region，3’-UTR），使靶蛋白mRNA降解或使其翻译受到抑制，从而调节内源基因的表达。miRNA参与了细胞增殖、迁移、凋亡、分化等多种细胞生命活动的调节。大量研究证实miRNA在几乎所有的恶性肿瘤中都存在表达异常，认为其在肿瘤发生发展中起到重要作用。近来研究认为其在肿瘤干细胞的自我更新、分化等过程中也扮演重要角色。根据miRNA在肿瘤细胞中的作用可将其分为两类，，一类在肿瘤细胞中表达升高，并且可通过抑制抑癌基因的表达促进肿瘤的发生，被称为促癌miRNA；另一类则其可通过抑制癌基因的表达抑制肿瘤的发生发展，被称为抑癌miRNA。作为研究较早的抑癌miRNA，miR-34a在多种肿瘤中发挥抑癌作用，近年来研究发现其在前列腺癌干细胞、胰腺癌干细胞中也发挥重要的调节作用，但其在乳腺癌干细胞中的作用及机制尚需进一步研究。 Notch信号通路是一个进化上相对保守的通路，在胚胎发育、器官成熟及肿瘤发生发展过程中均发挥重要的调控作用。Notch1是与乳腺癌的发生、转移及耐药密切相关的Notch受体之一。近年来研究发现其在维持乳腺癌干细胞的自我更新，调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可能在乳腺癌细胞及乳腺癌干细胞的调控中发挥重要作用。 本研究通过对临床组织样本的检测及体外实验，探讨了miR-34a靶向Notch1在乳腺癌细胞的增殖、侵袭迁移、乳腺癌干细胞调控及肿瘤耐药性中的作用，为乳腺癌的临床诊断及治疗提供了依据。 方法： 以qRT-PCR方法检测乳腺癌组织及正常组织中miR-34a及Notch1mRNA的表达水平，以免疫组化方法检测Notch1在乳腺癌中的表达水平，分析miR-34a与肿瘤转移、临床分期以及Notch1表达的相关关系。 以miR-34a模拟物、Notch1siRNA或其阴性对照分别转染MCF-7细胞，采用qRT-PCR及Western blot方法检测Notch1及其下游因子的表达变化，采用CCK-8法检测细胞增殖水平，采用Transwell法检测细胞侵袭及迁移的变化，Western blot法检测E-cadherin、Vimentin的表达变化。采用流式细胞术检测CD44+/CD24-细胞比例，Western blot法检测ALDH1表达水平变化。 构建表达Notch1胞内结构域的pcDNA3.1-Notch1质粒，并将其与miR-34a模拟物共转染于MCF-7细胞中，检测各组细胞增殖水平、迁移能力、CD44+/CD24-细胞比例及ALDH1的表达变化，以观察miR-34a/Notch1在乳腺癌细胞增殖、侵袭迁移以及乳腺癌干细胞调控中的作用。 将miR-34a模拟物与紫杉醇分别或联合作用于MCF-7细胞，以CCK-8法观察对细胞活力的影响，以乳腺球形成实验检测乳腺癌干细胞自我更新能力，以Western blot方法检测Notch1、Hes1、ABCG2及ALDH1的表达水平，探讨miR-34a影响MCF-7细胞化疗敏感性的作用机制。 结果： miR-34a在乳腺癌组织中的表达水平明显低于正常组织，在伴有淋巴结转移的乳腺癌组织中明显低于不伴淋巴结转移的乳腺癌组织。在临床Ⅰ-Ⅲ期患者中，乳腺癌组织中miR-34a表达水平随肿瘤分期的升高而降低。在乳腺癌组织中，miR-34a与Notch1的表达水平呈负相关。转染miR-34a模拟物上调miR-34a后可观察到Notch通路中Notch1及Hes1的表达水平明显下降。双荧光素酶报告基因分析显示miR-34a与Notch13’-UTR间存在直接作用的靶点。上调miR-34a能够明显抑制MCF-7细胞的增殖、侵袭及迁移水平，与下调Notch1的作用相似。而在过表达miR-34a的细胞中上调Notch1能够部分逆转miR-34a对乳腺癌细胞的抑制作用。提示miR-34a/Notch1在调节乳腺癌细胞增殖、侵袭及迁移过程中占有重要地位。对乳腺癌干细胞标志物ALDH1、CD44、CD24的检测发现，上调miR-34a及下调Notch1均能够明显抑制ALDH1的表达水平，降低CD44+/CD24-细胞比例，而在过表达miR-34a的细胞中同时上调Notch1则能够部分逆转miR-34a对乳腺癌干细胞的抑制作用。提示Notch1通路是介导miR-34a抑制乳腺癌干细胞作用的重要机制之一。将紫杉醇与miR-34a模拟物共同作用于MCF-7细胞后发现，miR-34a能够明显增加紫杉醇对细胞的抑制作用。与应用紫杉醇治疗相比，miR-34a模拟物及紫杉醇的联合应用能使乳腺球形成效率及Notch1、Hes1、ABCG2、ALDH1的表达水平均明显降低。提示miR-34增加紫杉醇药物敏感性的机制与其抑制Notch1通路及抑制乳腺癌自我更新能力有关。 结论： miR-34a能够通过靶向Notch1抑制乳腺癌细胞增殖、侵袭及迁移能力并抑制乳腺癌干细胞特性。同时miR-34a能够增加紫杉醇药物敏感性，其机制与抑制Notch1通路及抑制乳腺癌干细胞的自我更新能力有关。这些结果提示上调miR-34a的表达是增强乳腺癌疗效的有效途径。
[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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