TRIP4促进黑色素瘤生长并影响BRAF抑制剂敏感性

发布时间：2018-03-11 01:30

本文选题：TRIP4　切入点：黑色素瘤　出处：《大连医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:黑色素瘤作为一种恶性的皮肤肿瘤,放化疗的效果差且具有不良预后的情况,因此新的治疗靶点的发现仍然是亟待解决的问题。本研究利用SiRNA文库筛选技术,在黑色素瘤细胞系A375细胞中进行筛选,发现敲低甲状腺激素受体相互作用因子4(Thyroid Hormone Receptor Interactor 4,TRIP4)能够影响黑色素瘤细胞的生长,而该基因在黑色素瘤中的功能及相关机制的研究尚不明确。因此,我们在确定该基因在黑色素瘤细胞及病人组织中的表达水平后,选择不同黑色素瘤细胞系,敲低或过表达TRIP4之后,探索TRIP4对黑色素瘤生长的调控机制及生物学功能;并在裸鼠体外成瘤模型中进一步验证TRIP4调控黑色素瘤生长的的相关机制,深入研究TRIP4调控黑色素瘤生长的的分子机制并明确TRIP4的临床意义。本论文拟在此基础上确立TRIP4作为黑色素瘤治疗新靶点提供理论和实验依据。方法:(1)以siRNA library screen为实验新技术,寻找影响黑色素瘤细胞中的潜在靶基因。(2)应用基因转染技术来过表达或敲低TRIP4,研究其对细胞增殖、迁移、侵袭、凋亡等生物学功能,以及相关分子信号通路的变化。(3)应用免疫印迹、免疫组化等技术检测人肿瘤细胞和正常细胞以及组织芯片标本中TRIP4的表达水平,并结合细胞特性和临床资料分析TRIP4的肿瘤特异性表达和它们在预测临床预后上的价值。(4)应用Pull down方法及染色质免疫共沉淀(Ch IP)技术检测TRIP4与炎症因子COX-2/iNOS启动子区结合情况。(5)使用免疫共沉淀(Co-IP)技术检测TRIP4与p300的相互作用。(6)利用细胞活性实验检测TRIP4对BRAF抑制剂的影响。(7)建立黑色素瘤裸鼠动物模型,进一步验证TRIP4对肿瘤生长,对COX-2/iNOS表达以及药物敏感性的影响。结果:(1)在本实验中,我们通过siRNA library screen筛选鉴定出TRIP4基因,发现TRIP4在黑色素瘤细胞和组织中高表达。(2)敲低TRIP4能够抑制黑色素瘤细胞的生长。(3)通过体内和体外实验证明TRIP4靶向炎症因子调控黑色素瘤的生长。(4)TRIP4可以通过NF-κB通路调控黑色素瘤细胞中炎症因子COX-2和iNOS的表达。(5)TRIP4可直接与COX-2和iNOS的启动子区结合进一步调控二者的表达。(6)TRIP4可协同p300靶向炎症因子调控黑色素瘤细胞的生长进程。(7)体内和体外实验证明敲低TRIP4可增强BRAF抑制剂的抗黑色素瘤生长作用。(8)黑色素瘤病人组织中,TRIP4与COX-2和iNOS的表达成正相关,并与黑色素瘤病人的不良预后呈正相关。结论:TRIP4在黑色素瘤病人组织中具有高的表达水平,并与病人的不良预后呈正相关。进一步研究发现TRIP4能够协同p300靶向炎症因子调控黑色素瘤的生长进程,此外,我们还发现TRIP4能够影响黑色素瘤靶向药物的敏感性。综上所述,我们目前的研究结果为TRIP4作为一个潜在的预后标志物和治疗靶点提供了基础理论依据,而TRIP4和BRAF的双靶向作用为黑色素瘤治疗的临床策略提供了可能性。
[Abstract]:Objective: melanoma is a malignant skin tumor, radiotherapy and chemotherapy have poor effect and poor prognosis, so new therapeutic target discovery is still a problem to be solved. This study used SiRNA library screening technology, were screened in the melanoma cell line A375, found that knockdown of thyroid gland hormone receptor interaction factor 4 (Thyroid Hormone Receptor Interactor 4, TRIP4) can affect the growth of melanoma cells, and study the function of this gene in melanoma and its mechanism is not clear. Therefore, we determine the expression level of the gene in melanoma cells and tissues of patients. Choose different melanoma tumor cell lines, after knockdown or overexpression of TRIP4, to explore the regulatory mechanism and biological function of TRIP4 on the growth of melanoma; and to further verify the regulation of TRIP4 tumor model in nude mice in vitro The growth of melanoma related mechanisms, in-depth study of the clinical significance of TRIP4 regulates the growth of melanoma and clear the molecular mechanism of TRIP4. This thesis is based on the establishment of TRIP4 as a new target for the treatment of melanoma and provide theoretical and experimental basis. Methods: (1) siRNA library screen to new technology experiment. Looking for potential target genes in melanoma cells. (2) the application of gene transfection to expression or knockdown of TRIP4 on cell proliferation, migration, invasion, apoptosis and other biological functions, and the related molecular signaling pathway changes. (3) by Western blot, immunohistochemistry human tumor cells and normal cells and TRIP4 microarray expression levels, combined with the characteristics and clinical data analysis of tumor cell specific expression of TRIP4 and their roles in predicting the clinical prognosis value. (4) by Pull down Assay and chromatin immunoprecipitation (Ch IP) detection of TRIP4 and inflammatory factors in the promoter region of COX-2/iNOS. (5) combined with immunoprecipitation (Co-IP) interaction detection of TRIP4 and P300. (6) the effect of cell activity assay of TRIP4 BRAF inhibitors. (7) the establishment of melanin nude mice animal model, further validation of TRIP4 on tumor growth and influence on the expression of COX-2/iNOS and drug sensitivity. Results: (1) in this experiment, we through the siRNA library screen were identified in the TRIP4 gene, found high expression of TRIP4 in melanoma cells and tissues. (2) knockdown of TRIP4 inhibited black melanoma cell growth. (3) by in vivo and in vitro experiments show that TRIP4 target control to the inflammatory factor in the growth of melanoma. (4) TRIP4 by COX-2 and iNOS NF- expression of inflammatory factor kappa B pathway in melanoma cells. (5) TRIP4 The promoter region of COX-2 directly with the combination of iNOS and the expression of further regulation of the two. (6) TRIP4 can cooperate with P300 targeting inflammatory factors regulate melanoma cell growth process. (7) in vivo and in vitro experiments show that knockdown of TRIP4 can enhance the anti melanoma effect of BRAF inhibitor (8) black. Patients with melanoma tissues, the expression of TRIP4 and COX-2 and iNOS are positively correlated, and was related to the poor prognosis of melanoma patients. Conclusion: TRIP4 has high expression levels in patients with melanoma tissues, and was related to the poor prognosis of patients. Further study found that TRIP4 co P300 targeting inflammation the process of growth factor, regulation of melanoma in addition, we also found that TRIP4 can influence melanoma targeting drug sensitivity. In summary, we present results for TRIP4 as a potential prognostic marker and therapeutic target. The basic theoretical basis is provided, and the dual targeting effect of TRIP4 and BRAF provides a possibility for the clinical strategy of melanoma treatment.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.5

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