鼻咽癌中STING信号通路调控髓源性抑制细胞的分子机制及临床意义研究

发布时间：2018-02-14 04:10

本文关键词： 鼻咽癌 STING 髓源性抑制细胞 miR-24 预后　出处：《北京协和医学院》2016年博士论文　论文类型：学位论文

【摘要】：背景鼻咽癌(Nasopharyngeal carcinoma, NPC)是一种高发于我国华南地区的恶性肿瘤,95%以上的NPC与EB病毒(Epstein-Barr virus, EBV)感染密切相关。放化疗为基础的综合治疗是目前主流的治疗方式,但局部进展的NPC 5年复发率在40%以上,复发难治、远处转移的NPC中位总生存时间不足1年。肿瘤免疫治疗,因其低毒性和潜在的治愈性,成为当下研究的热点。PD-1抗体、过继性T细胞回输等免疫治疗,已在NPC显示出初步的疗效。然而,已有的免疫治疗有效率偏低：肿瘤微环境中的抑制性细胞、负向调控分子,是肿瘤免疫逃逸及免疫治疗失败的重要原因。髓源性抑制细胞(myeloid-deprived suppressor cells, MDSC),是一种具有抑制功能的未成熟髓系细胞。MDSC在NPC大量扩增、活化,抑制了杀伤性T细胞的功能,促进了肿瘤的侵袭和转移。STING信号通路,是免疫系统识别细胞质中异常来源的双链DNA,发挥天然免疫的重要通路：疱疹病毒、结核分枝杆菌等病原体的DNA,进入树突细胞的细胞质,该通路被激活,树突细胞合成大量的Ⅰ型干扰素,促进抗原呈递、启动适应性免疫反应。近两年的研究表明：来源于肿瘤的DNA,也可以激活STING信号通路,启动抗肿瘤免疫反应。实验目的研究STING信号通路在NPC的活化状态,探索该通路与MDSC分化的关系及临床意义。实验材料和方法1.检测STING信号通路的活化状态：利用免疫组化、ELSI A (the enzyme-linked immunosorbent assay)、Western blot等,检测临床样本(肿瘤及癌旁组织、患者及健康人血清)和细胞系(癌细胞系、正常鼻咽上皮细胞系)中STING的表达、信号通路的活性。2.分析STING信号通路影响MDSC的分化：利用过表达、siRNA沉默、磷酸化位点突变等技术手段以及癌细胞和CD33+细胞共培养体系,流式检测分析STING信号通路对MDSC分化的影响。收集共培养体系上清,ELISA测定IL-6、GM-CSF、IL-1β等细胞因子。3.筛查调控STING的miRNA:结合生物信息学预测,利用qPCR、Western Blot及荧光素酶报告基因实验,筛查并验证NPC中调控STING的miRNA.4.研究STING信号通路的临床意义：收集临床资料,统计分析STING信号通路与MDSC水平、EB病毒DNA载量、分期、预后等临床指标的相关性。实验结果1. STING信号通路在NPC中下调：癌组织中的STING表达量显著地低于配对的癌旁组织(n=22,p0.001),患者血清中的STING浓度显著地低于健康人(p=0.025)。癌细胞系(TW03、CNE2)中STING的表达量,低于正常鼻咽上皮细胞系(NP69); HSV-1刺激后,TW03、CNE2中p-TBK1、p-IRF3水平及IFN-β mRNA转录水平,低于NP69。2. STING信号通路的下调促进了MDSC的分化：相比于对照组,过表达STING的TW03,诱导产生的MDSC数目明显减少(p0.05),对T细胞增殖的抑制能力明显减弱(p0.05)；利用siRNA技术敲低TW03中的STING或将STING磷酸化位点突变,诱导MDSC的结果正好相反。患者肿瘤组织中,STING与CD33的表达量,呈显著的负相关(n=50,p0.001).3.miR-24调控STING的表达：生物信息学预测,STING是miR-24的直接作用靶点；TW03、CNE2细胞系中过表达miR-24,可下调STING mRNA的转录及STING蛋白的表达；荧光素酶报告基因实验证实,miR-24可直接结合STING 3'UTR。4.肿瘤组织中STING表达的临床意义：患者肿瘤组织中STING的表达量,与血清中STING的浓度线性相关(n=50,r=0.5928,P0.0001),与基线EB病毒DNA载量负相关(n=50,P=0.041),与接受同步放疗后的无复发生存时间正相关(n=89,p=0.041)。结论：STING信号通路在鼻咽癌中下调,促进了MDSC的分化,增强了MDSC的免疫抑制功能,是患者预后不良的因素之一。此外,miR-24在鼻咽癌中过表达,可能是STING表达下降的原因之一。
[Abstract]:The background of nasopharyngeal carcinoma (Nasopharyngeal carcinoma NPC) is in China's Southern China region has a high incidence of malignant tumors, NPC and EB more than 95% viruses (Epstein-Barr virus, EBV) infection is closely related. Comprehensive treatment based chemoradiotherapy is the main treatment method, but the Bureau of the Ministry of the progression of NPC 5 year recurrence rate in 40% above, relapsed, distant metastasis NPC median overall survival time of less than 1 years. Tumor immunotherapy, due to its low toxicity and potential cure, become the focus of current research of.PD-1 antibody, adoptive T cell transfusion immune therapy, has been in the NPC show preliminary curative effect. However, immunotherapy the effective rate is low: the microenvironment of the tumor suppressor cells, negative regulatory molecules, is an important reason for the failure of tumor immune escape and immunotherapy. Myeloid derived suppressor cells (myeloid-deprived, suppressor, cells, MDSC) is a kind of Immature myeloid cells, inhibition of.MDSC function in NPC amplification, activation, inhibition of cytotoxic T cells, promote tumor invasion and metastasis of.STING signaling pathway, double stranded DNA abnormal immune system recognition in the cytoplasm, important innate immune pathways play: herpes simplex virus, Mycobacterium tuberculosis and other pathogens DNA, enter the dendritic cell cytoplasm, the pathway is activated, dendritic cells synthesize large amounts of type I interferon, promoting antigen presentation, the initiation of adaptive immune responses. Nearly two years of study showed that tumor derived DNA can activate STING signaling pathway, start the antitumor immune response. The purpose of the STING signaling pathway in the activation of the state of NPC, and the clinical significance of the relationship between the MDSC pathway and the differentiation of exploration. The activation of the experimental materials and methods 1. detection of STING signal pathway by immunohistochemistry (A, ELSI The enzyme-linked immunosorbent assay), Western blot, detection of clinical samples (tumor and paracancerous tissue, serum of patients and healthy people) and cell lines (cancer cells, normal nasopharyngeal epithelial cell line) the expression of STING in.2. pathway analysis on the effect of STING signaling pathway of MDSC expression by siRNA differentiation: silence, the co culture system of phosphate mutation technology and cancer cells and CD33+ cells, flow cytometry analysis of effects of STING signal pathway on the differentiation of MDSC. Co culture system was collected, ELISA IL-6 GM-CSF, IL-1 assay, cell factor beta.3. screening regulation of STING miRNA: combined with bioinformatics prediction, using qPCR. Western Blot and luciferase reporter assay, screening and clinical significance of NPC STING in the miRNA.4. verification regulation of STING signal pathway: clinical data collection, statistical analysis of STING signal pathway With the level of MDSC, EB staging DNA virus load, and correlation of prognosis. The experimental results of 1. STING signaling pathway in NPC down-regulation: expression of STING in the cancer tissues was significantly lower than that in paired adjacent tissues (n=22, p0.001), the concentration of STING in serum of patients were significantly lower than those of healthy people (p=0.025). Cancer cell lines (TW03, CNE2) expression in STING, lower than that of normal nasopharyngeal epithelial cell line (NP69); after HSV-1 stimulation, TW03, CNE2 p-TBK1, p-IRF3 level and IFN- beta mRNA transcription level, lower than the down-regulation of NP69.2. STING signaling pathway promotes the differentiation of MDSC: compared to the control group. The expression of STING TW03, the number of MDSC induced significantly reduced (P0.05), inhibition on the proliferation of T cells was significantly decreased (P0.05); the knockdown of TW03 in STING or STING phosphorylation site mutation induced by siRNA technology, MDSC on the contrary. The tumor tissue of patients with In the expression of STING and CD33, a significant negative correlation (n=50, p0.001) regulates the expression of.3.miR-24 STING: bioinformatics prediction, STING is a direct target of miR-24; TW03, miR-24 overexpression in CNE2 cells, the expression of STING could be down regulated by the transcription of mRNA and STING protein; luciferase reporter gene the experiment confirmed that miR-24 can be directly combined with the clinical significance of the expression of STING STING 3'UTR.4. in tumor tissues: the expression of STING in tumor tissues of patients with the correlation is linear with the concentration of STING in serum (n=50, r=0.5928, P0.0001), and baseline EB virus DNA load negative correlation (n=50, P=0.041), is associated with recurrence the survival time of accept synchronization after radiotherapy (n=89, p=0.041). Conclusion: the down-regulation of STING signaling pathway in nasopharyngeal carcinoma, promote MDSC differentiation, enhance the immunosuppressive function of MDSC, is one of the factors of poor prognosis. In addition, miR-24 in the nose Overexpression in the cancer of the pharynx may be one of the reasons for the decline of STING expression.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.63

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