本文選題：腦膠質(zhì)瘤 + 替莫唑胺��； 參考：《中南大學(xué)》2014年博士論文

【摘要】：[研究背景] 腦膠質(zhì)瘤是最常見的顱內(nèi)腫瘤之一,侵襲性生長的生物學(xué)特性使其治療效果不盡理想。膠質(zhì)瘤的形成可能是一個多因素參與、多步驟累積的病理過程。因此,從發(fā)生發(fā)展機(jī)制入手,探尋腦膠質(zhì)瘤早期診斷和治療的分子靶點依然是目前神經(jīng)外科領(lǐng)域面臨的重要課題之一 miRNA是一類內(nèi)源性的非編碼RNA,其異常表達(dá)不僅與腦膠質(zhì)瘤的發(fā)生發(fā)展密切相關(guān),而且也為探尋腦膠質(zhì)瘤新的診斷和治療方法提供了全新的思路。我們前期構(gòu)建了WHO不同分化級別的腦膠質(zhì)瘤的差異miRNA表達(dá)譜和差異mRNA表達(dá)譜,并通過整合分析發(fā)現(xiàn)miR-107、miR-124、miR-149等可以調(diào)控多個靶基因的表達(dá),而Rap1B等基因又可受miR-128a/128b、miR-149、miR-181b等多個miRNA調(diào)控。通過芯片數(shù)據(jù)進(jìn)行Pearson相關(guān)性分析發(fā)現(xiàn),Rap1B與miR-128a/b、miR-149和miR-181b呈明顯負(fù)相關(guān),提示Rap1B可能受這些miRNA分子直接調(diào)控,參與腦膠質(zhì)瘤的發(fā)生和發(fā)展。 本研究擬在此基礎(chǔ)上進(jìn)一步證實Rap1B是miR-128、miR-149及miR-181的直接調(diào)控靶基因,并證實它們在不同WHO分化級別的星形膠質(zhì)細(xì)胞瘤中的表達(dá)變化,進(jìn)而闡明miR-128、miR-149及miR-181通過靶向Rap1B對腦膠質(zhì)瘤細(xì)胞增殖和侵襲的影響以及增強(qiáng)替莫唑胺(temozolomide, TMZ)對腦膠質(zhì)瘤細(xì)胞化療敏感性的作用機(jī)制。 [Rap1B是miR-128、miR-149和miR-181a/b/c/d共同作用靶基因] 通過targetScan、Picta和miRDB等軟件分析,均預(yù)測到Rap1B是miR-128、miR-149和miR-181a/b/c/d的共同靶基因。通過分別構(gòu)建含Rap1B和miR-128、miR-149和miR-181a/b/c/d不同結(jié)合位點的熒光素酶報告基因載體及突變體,熒光素酶活性分析、Real-time PCR和Western blotting等實驗證實了Rap1B是miR-128、miR-149和miR-181a,miR-181b, miR-181c和miR-181d的共同作用靶基因,miR-128、miR-149和miR-181家族各成員可以在轉(zhuǎn)錄及轉(zhuǎn)錄后水平調(diào)控Rap1B的表達(dá)。 [miR-128、miR-149和miR-181a/b/c/d及Rap1B在WHO不同分化級別星形膠質(zhì)細(xì)胞瘤中的表達(dá)及相關(guān)性分析] 我們收集了WHO不同分化級別的星形膠質(zhì)細(xì)胞瘤組織及正常對照腦組織標(biāo)本,通過Real-time PCR檢測了miR-128、miR-149和miR-181a/b/c/d及靶基因Rap1B在星形膠質(zhì)細(xì)胞瘤中的表達(dá)。我們的研究結(jié)果表明,miR-128和miR-181家族各分子的表達(dá)下調(diào)均與星形膠質(zhì)瘤細(xì)胞瘤的發(fā)生密切相關(guān),而miR-149的表達(dá)下調(diào)和Rap1B的表達(dá)上調(diào)與星形膠質(zhì)細(xì)胞瘤的發(fā)生及惡性級別的進(jìn)展密切相關(guān)。Pearson相關(guān)性分析證實了miR-128、miR-149和miR-181a/b/c/d與Rap1B在星形膠質(zhì)細(xì)胞瘤中的表達(dá)呈負(fù)相關(guān),這種負(fù)相關(guān)與WHO的分化級別進(jìn)展無關(guān)。 [miR-128、miR-149和miR-181a/b/c/d通過Rap1B對腦膠質(zhì)瘤細(xì)胞增殖和侵襲的影響] 研究表明,異常表達(dá)的miRNA分子通�？梢园l(fā)揮癌基因或者是抑癌基因的作用,參與腫瘤的發(fā)生發(fā)展。我們通過細(xì)胞克隆形成實驗、MTT和Transwell實驗分別檢測了轉(zhuǎn)染miR-128、miR-149和miR-181a/b/c/d等miRNAs mimics或干擾腦膠質(zhì)瘤細(xì)胞中內(nèi)源性Rap1B的表達(dá)后對腦膠質(zhì)瘤細(xì)胞克隆形成能力、增殖和侵襲能力的影響。結(jié)果表明,miR-128、miR-149和miR-181a/b/c/d的過表達(dá)和Rap1B的敲除可以抑制腦膠質(zhì)瘤細(xì)胞的增殖和侵襲能力,說明,miR-128、miR-149和miR-181a/b/c/d等多個miRNAs均可能作為抑瘤性miRNAs,通過作用其共同的靶基因Rap1B影響腦膠質(zhì)瘤細(xì)胞的侵襲性生物學(xué)行為。 [miR-128、miR-149和miR-181a/b/c/d增強(qiáng)腦膠質(zhì)瘤細(xì)胞化療敏感性的作用機(jī)制研究] TMZ是一種新型的腦膠質(zhì)瘤化療藥物,主要用于惡性腦膠質(zhì)瘤或者是復(fù)發(fā)性腦膠質(zhì)瘤的治療。盡管TMZ對惡性腦膠質(zhì)瘤的有效治療率達(dá)45%,但仍有相當(dāng)部分腦膠質(zhì)瘤對TMZ治療療效欠佳或逐漸出現(xiàn)繼發(fā)性耐藥現(xiàn)象�，F(xiàn)有的研究表明,miRNA作為潛在的腦膠質(zhì)瘤治療靶點,可增強(qiáng)TMZ對腦膠質(zhì)瘤細(xì)胞的化療敏感性。本課題進(jìn)一步的研究表明,miR-128、miR-149和]miR-181a/b/c/d的過表達(dá)均可以增加TMZ對腦膠質(zhì)瘤細(xì)胞的化療敏感性。Rap1B作為miR-128、miR-149和miR-181a/b/c/d的直接調(diào)控靶基因,是RAS樣小GTP結(jié)合蛋白家族成員Rap1的亞基,Rap1和Rho家族GTPases成員Racl、 RhoA和CDC42一樣,均可以參與調(diào)控細(xì)胞骨架重塑、細(xì)胞的粘附和侵襲等多種生物學(xué)行為。我們的研究也進(jìn)一步證實了TMZ可以通過上調(diào)miR-128、miR-149和miR-181家族分子的表達(dá),靶向抑制Rap1B的表達(dá),從而影響細(xì)胞粘附和細(xì)胞骨架相關(guān)蛋白CDC42、RhoA和N-cadherin的表達(dá),調(diào)控細(xì)胞骨架蛋白F-actin的表達(dá)和分布,抑制腦膠質(zhì)瘤細(xì)胞的侵襲性增殖。同時,]miR-128、miR-149和miR-181家族分子也可以通過靶向Rap1B介導(dǎo)的細(xì)胞骨架蛋白的重塑增強(qiáng)替莫唑胺對腦膠質(zhì)瘤細(xì)胞的化療敏感性。
[Abstract]:[research background]
Glioma is one of the most common intracranial tumors. The biological characteristics of invasive growth are not ideal for the treatment of glioma. The formation of glioma may be a multi factor participation and multi step cumulative pathological process. Therefore, the molecular target of the early diagnosis and treatment of glioma is still the present God from the development mechanism. One of the most important subjects in the field of surgery
MiRNA is a class of endogenous non coding RNA. Its abnormal expression is not only closely related to the development of glioma, but also provides a new way of thinking for the exploration of new diagnosis and treatment of glioma. We constructed the differential miRNA expression profiles and differential mRNA expression profiles of different differentiated gliomas of WHO. The integration analysis found that miR-107, miR-124, miR-149 and so on can regulate the expression of multiple target genes, while Rap1B and other genes can be regulated by miR-128a/128b, miR-149, miR-181b and so on. The Pearson correlation analysis of the chip data shows that Rap1B is negatively correlated with miR-128a/b, miR-149 and miR-181b, suggesting that these genes may be affected by these genes. Direct regulation of molecules is involved in the occurrence and development of gliomas.
On this basis, we further confirm that Rap1B is a direct target gene for miR-128, miR-149 and miR-181, and confirms their expression in astrocytoma at different WHO differentiation levels, and then elucidates the effect of miR-128, miR-149 and miR-181 on the proliferation and invasion of brain gelatoma cells through target Rap1B, and the enhancement of the enhancement. The mechanism of temozolomide (TMZ) on chemosensitivity of glioma cells.
[Rap1B is miR-128, miR-149 and miR-181a/b/c/d target genes together]
Through software analysis such as targetScan, Picta and miRDB, it is predicted that Rap1B is the common target gene of miR-128, miR-149 and miR-181a/b/c/d. By constructing the luciferase reporter gene carrier and mutant containing the different binding sites of Rap1B and miR-128, miR-149 and miR-181a/b/c/d, the luciferase activity analysis. The experiment confirmed that Rap1B is the common target gene of miR-128, miR-149 and miR-181a, miR-181b, miR-181c and miR-181d, and the members of miR-128, miR-149 and miR-181 family can regulate the Rap1B expression at transcriptional and post transcriptional levels.
Expression and correlation analysis of [miR-128, miR-149, miR-181a/b/c/d and Rap1B in different astrocytomas of WHO
We collected WHO different grade astrocytoma tissue and normal control brain tissue specimens. The expression of miR-128, miR-149, miR-181a/b/c/d and target gene Rap1B in astrocytoma was detected by Real-time PCR. Our results showed that the expression of each molecule in miR-128 and miR-181 family was down to star. The occurrence of glioma cell tumor is closely related, and the down regulation of miR-149 expression and the up regulation of Rap1B expression are closely related to the occurrence of astrocytoma and the progression of malignant grade,.Pearson correlation analysis confirms that miR-128, miR-149 and miR-181a/b/c/d and Rap1B are negatively correlated with the expression of Rap1B in astrocytoma, and this negative correlation and WHO The progression of differentiation is not related.
Effects of [miR-128, miR-149 and miR-181a/b/c/d on proliferation and invasion of glioma cells through Rap1B]
Studies have shown that abnormal expression of miRNA molecules can usually play the role of oncogene or tumor suppressor gene and participate in the development of tumor. We have developed cell clones and MTT and Transwell experiments to detect miRNAs mimics, such as miR-128, miR-149 and miR-181a/b/c/d, or interfere with endogenous Rap1B in glioma cells. The results showed that the overexpression of miR-128, miR-149 and miR-181a/b/c/d and the knockout of Rap1B could inhibit the proliferation and invasion of glioma cells, indicating that the multiple miRNAs of miR-128, miR-149 and miR-181a/ b/c/d may be used as tumor suppressor miRNAs. The interaction of its common target gene Rap1B affects invasive biological behavior of glioma cells.
Mechanisms of [miR-128, miR-149 and miR-181a/b/c/d enhancing chemosensitivity of glioma cells
TMZ is a new type of chemotherapeutic drug for glioma, which is mainly used for the treatment of malignant glioma or recurrent glioma. Although the effective rate of treatment for malignant glioma by TMZ is 45%, there are still a considerable number of gliomas that have poor curative effect on TMZ treatment or secondary drug resistance. The present study shows that miRNA is used as a method. The potential therapeutic target of glioma can enhance the chemosensitivity of TMZ to glioma cells. Further studies in this topic suggest that the overexpression of miR-128, miR-149 and]miR-181a/b/c/d can increase the chemosensitivity of TMZ to glioma cells,.Rap1B as a direct target gene for miR-128, miR-149 and miR-181a/b/c/d, and is RAS. The subunit of Rap1, a member of the small GTP binding protein family, Rap1 and the GTPases members of the Rho family, Racl, RhoA, and CDC42, are all involved in the regulation of cytoskeleton remodeling, cell adhesion and invasion. Our study also further confirmed that TMZ can be targeted by up regulation of miR-128, miR-149, and miR-181 family molecules. Inhibits the expression of Rap1B, which affects the expression of cell adhesion and cytoskeleton related proteins CDC42, RhoA and N-cadherin, regulates the expression and distribution of cytoskeleton F-actin, and inhibits the invasive proliferation of glioma cells. At the same time, the]miR-128, miR-149 and miR-181 family components can also be mediated by the target Rap1B mediated cytoskeleton protein. Remodeling enhances the chemosensitivity of temozolomide to glioma cells.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R739.41

【共引文獻(xiàn)】

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相關(guān)碩士學(xué)位論文 前10條

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