本文選題：miR-186 + 膠質(zhì)瘤��； 參考：《南京醫(yī)科大學(xué)》2017年博士論文

【摘要】：膠質(zhì)瘤是最常見的顱內(nèi)惡性腫瘤,基于其獨(dú)特的侵襲性生長(zhǎng)方式,目前的療效仍不理想。近年來,腫瘤的分子靶向治療發(fā)展迅猛,探尋膠質(zhì)瘤發(fā)生發(fā)展的分子生物學(xué)機(jī)制,發(fā)現(xiàn)新的有效治療靶點(diǎn)對(duì)攻克膠質(zhì)瘤這一頑疾具有重要意義。涉及RNA調(diào)控或蛋白水平的靶點(diǎn)研究始終是膠質(zhì)瘤生物治療領(lǐng)域的熱點(diǎn)。本課題就此分為兩部分,分別從MicroRNA和凋亡抑制蛋白入手開展相關(guān)研究。第一部分:miR-186靶向IGF-IR影響膠質(zhì)瘤增殖及侵襲能力的實(shí)驗(yàn)研究。MicroRNA(miRNA)是一類非編碼的短小RNA,目前發(fā)現(xiàn)其主要功能是在轉(zhuǎn)錄后階段觸發(fā)靶基因的降解或阻礙其翻譯。一系列研究證實(shí),表達(dá)異常的miRNA與各種腫瘤包括膠質(zhì)瘤有關(guān),miRNA可以作為抑癌基因或致癌基因參與腫瘤的發(fā)生發(fā)展。miRNA表達(dá)譜研究已經(jīng)證實(shí)miR-186在多種腫瘤中表達(dá)下調(diào),然而其在膠質(zhì)瘤發(fā)生發(fā)展過程中的潛在功能及相關(guān)機(jī)制仍未闡明。胰島素樣生長(zhǎng)因子Ⅰ受體(IGF-IR)是一種跨膜蛋白,是酪氨酸蛋白激酶類受體家族的重要成員。IGF-IR在多種惡性腫瘤中均有陽性表達(dá),其可能的效應(yīng)包括抑制細(xì)胞凋亡,誘導(dǎo)細(xì)胞異常增殖等,在疾病的發(fā)展進(jìn)程中發(fā)揮重要作用。本課題旨在檢測(cè)miR-186及IGF-IR在膠質(zhì)瘤中表達(dá)水平及其效應(yīng)并探討潛在的分子機(jī)制。首先通過RT-qPCR檢測(cè)miR-186的表達(dá)情況;構(gòu)建miR-186過表達(dá)載體并轉(zhuǎn)染至膠質(zhì)瘤細(xì)胞系;MTT法和細(xì)胞侵襲試驗(yàn)(Transwell法)分別檢測(cè)細(xì)胞增殖和侵襲。隨后通過生物信息學(xué)軟件分析判斷IGF-IR為miR-186新的靶基因,雙熒光素酶報(bào)告基因技術(shù)予以證實(shí)。同樣通過RT-qPCR檢測(cè)IGF-IR的表達(dá)情況并與miR-186進(jìn)行spearman相關(guān)性分析;通過熒光定量RT-qPCR法和免疫印跡法(Western blot)檢測(cè)轉(zhuǎn)染細(xì)胞的IGF-1R基因mRNA和蛋白含量。最后用基因沉默技術(shù)(siRNA)敲低IGF-1R基因表達(dá),RT-qPCR和Western blot法分別檢測(cè)RNA水平和蛋白水平的表達(dá),MTT法和Transwell法檢測(cè)細(xì)胞增殖及侵襲。研究結(jié)果表明,miR-186在腦膠質(zhì)瘤組織和細(xì)胞中顯著下調(diào),miR-186過表達(dá)可以抑制細(xì)胞的增殖和侵襲。IGF-1R證實(shí)為miR-186的直接靶基因,并且IGF-1RmRNA在膠質(zhì)瘤中呈現(xiàn)高表達(dá)并與miR-186呈負(fù)相關(guān)。此外,IGF-1R基因沉默可以抑制膠質(zhì)瘤細(xì)胞增殖和侵襲能力,與過表達(dá)miR-186的效應(yīng)類似。通過上述研究,我們認(rèn)為,miR-186通過靶向IGF-1R發(fā)揮膠質(zhì)瘤抑瘤因子作用,提示miR-186可以成為治療膠質(zhì)瘤的一個(gè)潛在靶點(diǎn)。第二部分:Apollon在膠質(zhì)瘤中過表達(dá)及影響細(xì)胞增殖和疾病預(yù)后的研究目前研究表明,惡性腫瘤的發(fā)生發(fā)展與細(xì)胞凋亡失衡有關(guān)。作為凋亡蛋白抑制劑家族中分子量最大的成員,Apollon被報(bào)道在多種人類腫瘤中發(fā)揮致癌作用。本課題的目的是探討Apollon異常表達(dá)對(duì)膠質(zhì)瘤患者的臨床影響以及對(duì)膠質(zhì)瘤細(xì)胞的效應(yīng)。首先通過免疫組化方法檢測(cè)Apollon在人腦膠質(zhì)瘤標(biāo)本及對(duì)照標(biāo)本中HE染色情況,分析Apollon表達(dá)量與患者臨床病理特征及患者的生存指標(biāo)的相關(guān)性。其后使用Apollon特異性小干擾RNA轉(zhuǎn)染膠質(zhì)瘤細(xì)胞,Western blot檢測(cè)Apollon蛋白表達(dá),cck-8法及transwell法檢測(cè)膠質(zhì)瘤細(xì)胞的遷移和侵襲。結(jié)果表明:Apollon蛋白主要在膠質(zhì)瘤細(xì)胞質(zhì)中陽性表達(dá),在正常腦組織中幾乎不表達(dá)。統(tǒng)計(jì)學(xué)分析顯示,Apollon蛋白在膠質(zhì)瘤中的免疫反應(yīng)評(píng)分(IRS)顯著高于對(duì)照的非腫瘤腦組織,并且高IRS患者具有更高的WHO分級(jí)和更短的總生存時(shí)間。進(jìn)一步的多因素分析顯示Apollon高表達(dá)是提示膠質(zhì)瘤患者不良預(yù)后的獨(dú)立因素。體外實(shí)驗(yàn)表明,沉默Apollon基因從而降低其蛋白表達(dá)可以有效抑制膠質(zhì)瘤細(xì)胞增殖和侵襲。根據(jù)以上研究結(jié)果,我們得出結(jié)論:Apollon失調(diào)可能與人腦膠質(zhì)瘤的發(fā)生及其病理改變相關(guān)聯(lián),極有可能是一個(gè)潛在的膠質(zhì)瘤預(yù)后標(biāo)志物和新的治療靶點(diǎn)。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.41

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