MiR-186、Apollon影响人胶质瘤细胞增殖和侵袭的研究

发布时间：2018-07-06 17:41

本文选题：miR-186 + 胶质瘤　；参考：《南京医科大学》2017年博士论文

【摘要】：胶质瘤是最常见的颅内恶性肿瘤,基于其独特的侵袭性生长方式,目前的疗效仍不理想。近年来,肿瘤的分子靶向治疗发展迅猛,探寻胶质瘤发生发展的分子生物学机制,发现新的有效治疗靶点对攻克胶质瘤这一顽疾具有重要意义。涉及RNA调控或蛋白水平的靶点研究始终是胶质瘤生物治疗领域的热点。本课题就此分为两部分,分别从MicroRNA和凋亡抑制蛋白入手开展相关研究。第一部分:miR-186靶向IGF-IR影响胶质瘤增殖及侵袭能力的实验研究。MicroRNA(miRNA)是一类非编码的短小RNA,目前发现其主要功能是在转录后阶段触发靶基因的降解或阻碍其翻译。一系列研究证实,表达异常的miRNA与各种肿瘤包括胶质瘤有关,miRNA可以作为抑癌基因或致癌基因参与肿瘤的发生发展。miRNA表达谱研究已经证实miR-186在多种肿瘤中表达下调,然而其在胶质瘤发生发展过程中的潜在功能及相关机制仍未阐明。胰岛素样生长因子Ⅰ受体(IGF-IR)是一种跨膜蛋白,是酪氨酸蛋白激酶类受体家族的重要成员。IGF-IR在多种恶性肿瘤中均有阳性表达,其可能的效应包括抑制细胞凋亡,诱导细胞异常增殖等,在疾病的发展进程中发挥重要作用。本课题旨在检测miR-186及IGF-IR在胶质瘤中表达水平及其效应并探讨潜在的分子机制。首先通过RT-qPCR检测miR-186的表达情况;构建miR-186过表达载体并转染至胶质瘤细胞系;MTT法和细胞侵袭试验(Transwell法)分别检测细胞增殖和侵袭。随后通过生物信息学软件分析判断IGF-IR为miR-186新的靶基因,双荧光素酶报告基因技术予以证实。同样通过RT-qPCR检测IGF-IR的表达情况并与miR-186进行spearman相关性分析;通过荧光定量RT-qPCR法和免疫印迹法(Western blot)检测转染细胞的IGF-1R基因mRNA和蛋白含量。最后用基因沉默技术(siRNA)敲低IGF-1R基因表达,RT-qPCR和Western blot法分别检测RNA水平和蛋白水平的表达,MTT法和Transwell法检测细胞增殖及侵袭。研究结果表明,miR-186在脑胶质瘤组织和细胞中显著下调,miR-186过表达可以抑制细胞的增殖和侵袭。IGF-1R证实为miR-186的直接靶基因,并且IGF-1RmRNA在胶质瘤中呈现高表达并与miR-186呈负相关。此外,IGF-1R基因沉默可以抑制胶质瘤细胞增殖和侵袭能力,与过表达miR-186的效应类似。通过上述研究,我们认为,miR-186通过靶向IGF-1R发挥胶质瘤抑瘤因子作用,提示miR-186可以成为治疗胶质瘤的一个潜在靶点。第二部分:Apollon在胶质瘤中过表达及影响细胞增殖和疾病预后的研究目前研究表明,恶性肿瘤的发生发展与细胞凋亡失衡有关。作为凋亡蛋白抑制剂家族中分子量最大的成员,Apollon被报道在多种人类肿瘤中发挥致癌作用。本课题的目的是探讨Apollon异常表达对胶质瘤患者的临床影响以及对胶质瘤细胞的效应。首先通过免疫组化方法检测Apollon在人脑胶质瘤标本及对照标本中HE染色情况,分析Apollon表达量与患者临床病理特征及患者的生存指标的相关性。其后使用Apollon特异性小干扰RNA转染胶质瘤细胞,Western blot检测Apollon蛋白表达,cck-8法及transwell法检测胶质瘤细胞的迁移和侵袭。结果表明:Apollon蛋白主要在胶质瘤细胞质中阳性表达,在正常脑组织中几乎不表达。统计学分析显示,Apollon蛋白在胶质瘤中的免疫反应评分(IRS)显著高于对照的非肿瘤脑组织,并且高IRS患者具有更高的WHO分级和更短的总生存时间。进一步的多因素分析显示Apollon高表达是提示胶质瘤患者不良预后的独立因素。体外实验表明,沉默Apollon基因从而降低其蛋白表达可以有效抑制胶质瘤细胞增殖和侵袭。根据以上研究结果,我们得出结论:Apollon失调可能与人脑胶质瘤的发生及其病理改变相关联,极有可能是一个潜在的胶质瘤预后标志物和新的治疗靶点。
[Abstract]:Glioma is the most common intracranial malignant tumor. Based on its unique invasive growth mode, the current curative effect is still not ideal. In recent years, the molecular targeting therapy of tumor has developed rapidly to explore the molecular biological mechanism of glioma development. It is found that new effective therapeutic targets are of great significance to the attack of glioma, which involves R. The target research of NA regulation or protein level has always been a hot spot in the field of biological therapy for glioma. This topic is divided into two parts. The first part: the experimental study on the effect of miR-186 targeting IGF-IR on the proliferation and invasion of glioma,.MicroRNA (miRNA) is a class of non coding. Short RNA, the main function of which is to trigger the degradation of the target gene at the post transcriptional stage or to prevent its translation. A series of studies have confirmed that the abnormal expression of miRNA is associated with a variety of tumors including glioma, and miRNA can be used as a tumor suppressor gene or oncogene in the occurrence of.MiRNA expression profiles in the tumor, which has confirmed that miR-186 is more than The potential function and related mechanisms of the tumor in the development of glioma have not been elucidated. The insulin like growth factor I receptor (IGF-IR) is a transmembrane protein. It is an important member of the tyrosine kinase receptor family,.IGF-IR, which is positive in various malignant tumors, and its possible effect package The purpose of this study is to detect the expression level and effect of miR-186 and IGF-IR in glioma and to explore the potential molecular mechanism. Firstly, the expression of miR-186 was detected by RT-qPCR, and the miR-186 overexpression vector was constructed and transfected to glia. The tumor cell line, MTT method and cell invasion test (Transwell method) were used to detect cell proliferation and invasion respectively. Then, IGF-IR was identified as a new target gene for miR-186 by bioinformatics software. Double luciferase reporter gene technique was confirmed. The expression of IGF-IR was detected by RT-qPCR and Spearman correlation analysis was carried out with miR-186. The IGF-1R gene mRNA and protein content of transfected cells were detected by fluorescence quantitative RT-qPCR and immunoblotting (Western blot). Finally, the expression of IGF-1R gene was knocked down by gene silencing technique (siRNA). The expression of RNA level and protein level was detected by RT-qPCR and Western blot respectively. The proliferation and invasion of cells were detected by MTT method and method. The results showed that miR-186 was significantly down regulated in the tissues and cells of glioma. MiR-186 overexpression could inhibit cell proliferation and invasion of.IGF-1R as a direct target gene for miR-186, and IGF-1RmRNA was highly expressed in glioma and negatively correlated with miR-186. In addition, IGF-1R gene silencing could inhibit the proliferation and invasion of glioma cells. Ability, similar to the effect of overexpressing miR-186. Through the above study, we believe that miR-186 can play glioma tumor suppressor by targeting IGF-1R, suggesting that miR-186 can be a potential target for the treatment of glioma. Second part: the study of Apollon over expression in glioma and the study of cell proliferation and disease prognosis The development of malignant tumor is related to the imbalance of cell apoptosis. As the largest member of the apoptosis protein inhibitor family, Apollon has been reported to play a carcinogenic role in a variety of human tumors. The purpose of this study is to explore the effect of abnormal expression of Apollon on patients with glioma and the effect on glioma cells. The immunohistochemical method was used to detect the HE staining of Apollon in human glioma and control specimens. The correlation between the expression of Apollon and the clinicopathological features of the patients and the survival index of the patients was analyzed. Then the Apollon specific small interference RNA was used to transfect glioma cells, Western blot was used to detect the expression of Apollon protein, CCK-8 method and transwel. The L method was used to detect the migration and invasion of glioma cells. The results showed that Apollon protein was mainly expressed in the cytoplasm of glioma and almost did not express in normal brain tissue. The statistical analysis showed that the immune response score of Apollon protein in glioma (IRS) was significantly higher than that of the non tumor brain tissue of the control, and the high IRS patients had higher levels. WHO classification and shorter total survival time. Further multivariate analysis showed that high expression of Apollon was an independent factor for the poor prognosis of glioma patients. In vitro experiments showed that silencing of Apollon gene and reducing its protein expression could effectively inhibit the proliferation and invasion of glioma cells. According to the results of the above study, we concluded that Apo Llon imbalance may be associated with the occurrence and pathological changes of human glioma. It is likely to be a potential prognostic marker and a new therapeutic target for glioma.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.41

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