下调NRARP基因对甲状腺未分化癌肿瘤学特性影响的研究

发布时间：2018-05-14 17:48

本文选题：Notch调节锚蛋白 + 甲状腺未分化癌　；参考：《第二军医大学》2016年博士论文

【摘要】：背景与目的甲状腺未分化癌(anaplastic thyroid cancer,ATC)约占甲状腺癌7%~8%,恶性程度极高,发展迅速,浸润性生长,早期转移,多数患者在确诊时已经出现了远处转移,与分化较好的甲状腺乳头状癌和滤泡状癌相比预后较差,一经诊断生存期通常一年左右。目前未分化癌尚无有效的治疗方法,治疗方式亦无统一标准。不管是手术治疗,还是放疗、化疗,单独或联合应用治疗甲状腺未分化癌都难以获得较好疗效。甲状腺未分化癌的治疗仍然是世界各医疗中心所面临的挑战,尽管进行了手术、放疗、化疗等各项临床治疗相关的研究,但目前为止未找到统一、有效的治疗方法。由于疾病发生率低,几乎所有有关甲状腺未分化癌的治疗结果都是回顾性的小样本资料,缺乏大样本前瞻性治疗方法的总结。当今随着分子生物学技术及免疫学技术的迅猛发展和人类对恶性肿瘤的发展机制认识不断深入,生物医学疗法已经成为第四种肿瘤的治疗模式,其中通过制定个体化的肿瘤靶向治疗,及其相应靶点检测,提高肿瘤的治疗效果,具有关键的临床意义。基于甲状腺未分化癌单纯手术、放疗、化疗疗效差的特点,各个肿瘤中心探索治疗未分化癌的新方法,其中甲状腺未分化肿瘤的靶向治疗为提高患者预后提供了新的思路。Notch信号在肿瘤的发生和发展过程中起着非常关键的作用,Notch信号紊乱不仅能够直接导致恶性肿瘤的发生,而且通过与其他信号通路的相互作用,可以间接地诱导肿瘤的最终形成。在很多恶性肿瘤的发生发展过程中均报道Notch信号通路的异常信号表达,如前列腺癌、子宫颈癌、乳腺癌等。Notch信号通路成员存在于甲状腺组织中,且Notch受体表达受促甲状腺激素的调节。甲状腺癌中Notch信号通路存在异常表达,如在乳头状癌和滤泡状癌中,Notch基因表达较正常甲状腺组织及癌旁组织高。Notch调节锚蛋白(NOTCH-regulated ankyrin repeat protein,NRARP)是Notch信号通路的重要因子。可能参与甲状腺未分化癌的发生发展。NRARP基因编码114氨基酸的残基蛋白,有两个锚蛋白重复序列,是Notch信号通路上的一个成员。NRARP可以阻碍Notch诱导的CBF-1活化,能促进胞内段降解,抑制Notch信号通路激活。NRARP表达受Notch基因调控,Notch基因表达升高可以诱导NRARP表达增高,说明NRARP作为机体的一种反馈机制,可限制Notch信号过度增强和持续表达。至今尚无关于notch基因和nrarp基因在甲状腺未分化癌的细胞和组织表达的临床及实验报道。本课题探讨nrarp基因在甲状腺未分化癌组织和细胞中的表达情况,并评估其对甲状腺未分化癌细胞增殖、凋亡、周期、迁移和侵袭的作用,为确定治疗未分化癌的关键靶点提供实验依据。方法收集甲状腺未分化癌标本34例,采用免疫组化方法检测并比较nrarp蛋白在癌组织和癌旁组织中的表达水平,并根据表达水平分成高表达和低表达组,进行预后生存分析;采用携带nrarp-shrna的慢病毒(lenti-nrarp-shrna)抑制nrarp基因mrna和蛋白的表达水平并进行验证;采用wst-1方法检测lenti-nrarp-shrna对甲状腺癌细胞株bht-101和8305c体外增殖的影响,并通过裸鼠荷瘤模型检测lenti-nrarp-shrna对细胞株体内增殖的影响;采用细胞流式仪技术检测lenti-nrarp-shrna对细胞株周期和凋亡的影响;采用transwell法检测lenti-nrarp-shrna对细胞迁移和侵袭的影响;采用westernblot技术检测lenti-nrarp-shrna处理后一系列与周期蛋白(p21、cyclind1)、凋亡蛋白(bax、bcl-2、caspase-3)、迁移调控有关蛋白的表达水平。结果nrarp蛋白在甲状腺癌组织中表达水平显著高于正常组织,lenti-nrarp-shrna显著抑制bht-101和8305c细胞体内和体外增殖;lenti-nrarp-shrna诱导p21蛋白表达、抑制cyclind1蛋白表达进而诱导细胞发生g1期阻滞;lenti-nrarp-shrna诱导bax蛋白表达、抑制bcl-2蛋白表达,并激活caspase-3蛋白,导致细胞凋亡;lenti-nrarp-shrna抑制mmp-9蛋白表达,抑制细胞迁移和侵袭。结果一、notch蛋白和nrarp蛋白在甲状腺癌组织及转移灶中较癌旁组织高表达。nrarp高表达与患者预后呈显著负性相关,提示nrarp可作为甲状腺癌患者靶向治疗的潜在靶点。二、干扰nrarp表达可显著抑制细胞株体内和体外增殖;可提高肿瘤周期相关抑癌基因p21表达,抑制促癌基因cyclind1,从而诱导肿瘤细胞g1期阻滞;使凋亡相关bax蛋白表达增高,bcl-2表达降低,caspase-3被激活,从而提高凋亡细胞比例;并抑制mmp-9表达进而减弱未分化癌细胞的侵袭能力。结论NRARP与甲状腺未分化癌发生发展密切相关,降低其表达水平可以诱导周期阻滞、促进细胞凋亡进而抑制甲状腺癌细胞迁移、增殖和侵袭,可成为甲状腺未分化癌靶向治疗的潜在靶点。
[Abstract]:Background and objective anaplastic thyroid cancer (ATC) accounts for about 7%~8% of thyroid cancer. It has a high degree of malignancy, rapid development, invasive growth and early metastasis. Most patients have had distant metastases at the time of diagnosis. Compared with the poorly differentiated thyroid papillary and follicular carcinomas, the prognosis is poor. It is usually about a year or so. There is no effective treatment for undifferentiated carcinoma and there is no unified standard for treatment. It is difficult to achieve better curative effect, whether it is surgical treatment, radiotherapy, chemotherapy, alone or combined with treatment of undifferentiated thyroid cancer. The treatment of undifferentiated thyroid cancer is still a challenge to various medical centers in the world, though Studies related to surgery, radiotherapy, chemotherapy and other clinical treatments were carried out, but the unified and effective treatment has not been found so far. Due to the low incidence of disease, almost all the results of the treatment of undifferentiated thyroid cancer are retrospective and small sample data, lack of a summary of large sample prospective treatment methods. The rapid development of physical and immunological technology and the deepening of human understanding of the development mechanism of malignant tumor. Biomedical therapy has become the treatment mode of fourth kinds of tumor. It is of key clinical significance to improve the therapeutic effect of tumor by formulating individualized tumor targeting therapy and its corresponding target detection. A new approach to the treatment of undifferentiated cancer in the center of the thyroid undifferentiated carcinoma with simple surgery, radiotherapy and chemotherapy, and the targeted treatment of undifferentiated thyroid tumors provides a new way of thinking to improve the prognosis of the patients. The.Notch signal plays a very important role in the occurrence and development of the tumor, Notch signal. The disorder can not only directly lead to the occurrence of malignant tumors, but also indirectly induce the final formation of the tumor by interaction with other signaling pathways. In the course of the development of many malignant tumors, the abnormal signal expression of Notch signaling pathway, such as prostate cancer, cervical cancer, breast cancer and other.Notch signaling pathways, is reported. In thyroid tissue, the expression of Notch receptor is regulated by thyroid stimulating hormone. There is an abnormal expression of Notch signaling pathway in thyroid carcinoma, such as in papillary and follicular carcinomas, Notch gene expression is higher than normal thyroid tissue and paracancerous tissue.Notch regulated anchorage white (NOTCH-regulated ankyrin repeat protein, NRARP). The important factor of Notch signaling pathway may be involved in the development and development of the.NRARP gene encoding 114 amino acid residues in the undifferentiated thyroid carcinoma, and there are two anchorage repeat sequences. A member of the Notch signaling pathway,.NRARP, can impede the activation of CBF-1 induced by Notch, promote the degradation of the intracellular segments and inhibit the activation of.NRAR by the Notch signaling pathway. The expression of P is regulated by Notch gene, and the increase of Notch gene expression can induce the increase of NRARP expression. As a feedback mechanism of the body, NRARP can restrict the excessive enhancement and continuous expression of Notch signal. There is no clinical and experimental report on the expression of Notch gene and nrarp gene in undifferentiated thyroid carcinoma. To investigate the expression of nrarp gene in the tissues and cells of undifferentiated thyroid carcinoma and evaluate its effect on the proliferation, apoptosis, cycle, migration and invasion of undifferentiated thyroid cancer cells, and provide experimental basis for determining the key targets for undifferentiated carcinoma. Methods 34 specimens of undifferentiated thyroid carcinoma were collected and detected by immunohistochemical method. The expression level of nrarp protein in cancer tissue and para cancer tissue was compared, and the expression level was divided into high expression and low expression group according to the expression level, and the prognosis survival analysis was carried out. The expression level of nrarp gene mRNA and protein was inhibited by the lentivirus (lenti-nrarp-shrna) carrying nrarp-shrna and the WST-1 method was used to detect lenti-nrarp-shrna. The effects of bht-101 and 8305c on the proliferation of thyroid cancer cell lines in vitro, and the effect of lenti-nrarp-shrna on the proliferation of cell lines in nude mice were detected by the nude mouse model. The effect of lenti-nrarp-shrna on cell cycle and apoptosis was detected by cell flow cytometry, and lenti-nrarp-shrna was used to detect the migration and invasion of cells by the Transwell method. Westernblot technique was used to detect the expression level of protein in lenti-nrarp-shrna, p21, CyclinD1, apoptosis protein (Bax, Bcl-2, caspase-3), and the expression level of nrarp protein in thyroid carcinoma tissue was significantly higher than that of normal tissue, lenti-nrarp-shrna significantly inhibited bht-101 and 8305. The proliferation of C cells in vivo and in vitro, lenti-nrarp-shrna induced p21 protein expression, inhibition of cyclinD1 protein expression and induced G1 phase block, lenti-nrarp-shrna induced Bax protein expression, inhibition of Bcl-2 protein expression, and activation of caspase-3 protein, leading to cell apoptosis; lenti-nrarp-shrna inhibits the expression of MMP-9 protein, inhibits cell migration and inhibits cell migration. The high expression of Notch protein and nrarp protein in thyroid carcinoma tissues and metastatic foci was significantly negatively correlated with the prognosis of the patients, suggesting that nrarp could be a potential target for the target treatment of thyroid cancer patients. Two, interference nrarp expression could inhibit the proliferation of cell lines in vivo and in vitro, and improve the swelling of the cells. The expression of tumor suppressor gene p21, inhibiting the oncogene CyclinD1, inducing the G1 block of tumor cells, increasing the expression of Bax protein, decreasing the expression of Bcl-2, activating the Caspase-3, and increasing the percentage of apoptotic cells, and inhibiting the MMP-9 expression to weaken the invasion ability of undifferentiated cancer cells. Conclusion NRARP and thyroid are not. It is closely related to the development and development of differentiated cancer. Reducing its expression level can induce cell cycle arrest, promote cell apoptosis and inhibit the migration, proliferation and invasion of thyroid cancer cells, which may be a potential target for targeted therapy for undifferentiated thyroid cancer.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R736.1

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