本文選題：miR-92a + 腦膠質(zhì)瘤�。� 參考：《東南大學(xué)》2017年博士論文

【摘要】：腦膠質(zhì)瘤是中樞神經(jīng)系統(tǒng)中最常見的原發(fā)性腫瘤之一,惡性水平高,具有很高的發(fā)病率與死亡率。近年來,雖然人們在腦膠質(zhì)瘤的手術(shù)醫(yī)治以及術(shù)后放療、化療等方面取得了長足的進(jìn)步,但是惡性腦膠質(zhì)瘤病人的預(yù)后依然很差,確診后的平均生存周期不超過15個月。最新的研究發(fā)現(xiàn),腦膠質(zhì)瘤細(xì)胞中存在一小部分具備干細(xì)胞特征的細(xì)胞亞群,這類細(xì)胞可以持續(xù)增殖,具備自我更新能力和多向分化潛能,被定義為腦膠質(zhì)瘤干細(xì)胞(Glioma stem cells, GSCs)。GSCs細(xì)胞的存在是腦膠質(zhì)瘤形成、浸潤、轉(zhuǎn)移、復(fù)發(fā)以及放化療耐受的主要原因。MicroRNAs (miRNAs)是一類在進(jìn)化上高度保守,長度大約在22nt左右的非編碼RNA分子,可以通過直接作用于下游mRNAs的3端非翻譯區(qū)(3'Untranslated Regions,3'UTR)阻止mRNAs的轉(zhuǎn)錄,和域誘導(dǎo)mRNAs的降解,進(jìn)而在轉(zhuǎn)錄后水平調(diào)控機體內(nèi)基因的表達(dá)。越來越多的證據(jù)表明,miRNAs分子能夠起著類似于原癌基因或者抑癌基因的作用,影響腫瘤細(xì)胞的形成與發(fā)展。此外,也有文獻(xiàn)報道指出miRNAs在腫瘤干細(xì)胞分化和自我更新的調(diào)控中也發(fā)揮著重要的功能。因此,可以通過改變miRNAs的表達(dá)量來降低腫瘤細(xì)胞和腫瘤干細(xì)胞的惡性程度,這可能是一個很有前景的治療方案。在本研究中,作者通過高通量測序技術(shù)篩查發(fā)現(xiàn),與腦膠質(zhì)瘤細(xì)胞相比,GSCs細(xì)胞中miR-92a的表達(dá)水平顯著降低。之前的文獻(xiàn)報道表明,miR-92a在腦膠質(zhì)瘤細(xì)胞系和腦膠質(zhì)瘤病人的組織標(biāo)本中高表達(dá),降低miR-92a的表達(dá)水平可以抑制腦膠質(zhì)瘤細(xì)胞的增殖并促進(jìn)細(xì)胞凋亡。然而,miR-92a在腦膠質(zhì)瘤細(xì)胞遷移、侵襲以及GSCs細(xì)胞生存、遷移和自我更新中的功能尚不明確。所以,充分了解miR-92a在這兩種細(xì)胞中的功能和具體分子機制,可以為腦膠質(zhì)瘤的治療提供新的思路。研究目的探索miR-92a在腦膠質(zhì)瘤以及GSCs細(xì)胞中的功能和詳細(xì)作用機制,為腦膠質(zhì)瘤的靶向治療提供新的理論依據(jù)。實驗方法1.采用無血清饑餓培養(yǎng)法從腦膠質(zhì)瘤細(xì)胞(U87和U251)中分離培養(yǎng)出GSCs細(xì)胞,并用免疫熒光染色法鑒定GSCs細(xì)胞;2.采用第二代高通量測序技術(shù)比較U87細(xì)胞和來源于U87的GSCs細(xì)胞U87s中miRNAs的表達(dá)量差異;3.采用MTT、細(xì)胞劃痕、Transwell以及Annexin V-FITC/PI雙染法檢測miR-92a對U87和U251細(xì)胞體外增殖、遷移、侵襲和凋亡的影響;4.采用裸鼠成瘤實驗檢測miR-92a在體內(nèi)對腦膠質(zhì)瘤細(xì)胞生長的影響;5.采用qPCR、Western Blot和腫瘤干細(xì)胞球成球等實驗檢測miR-92a對GSCs細(xì)胞生長、遷移以及自我更新的影響;6.結(jié)合生物信息學(xué)方法和雙熒光素酶報告系統(tǒng)預(yù)測并鑒定miR-92a在腦膠質(zhì)瘤細(xì)胞和GSCs細(xì)胞中的靶基因;7.采用Western Blot和免疫熒光染色法確定miR-92a在腦膠質(zhì)瘤細(xì)胞和GSCs細(xì)胞中作用的分子機制。實驗結(jié)果1.利用無血清饑餓培養(yǎng)法從腦膠質(zhì)瘤細(xì)胞(U87和U251)中分離出GSCs細(xì)胞(U87s和U251s),免疫熒光染色實驗結(jié)果表明,GSCs細(xì)胞表面表達(dá)腫瘤干細(xì)胞的分子標(biāo)志CD133和神經(jīng)干細(xì)胞的分子標(biāo)志Nestin,分化后的GSCs細(xì)胞表達(dá)神經(jīng)膠質(zhì)細(xì)胞的分子標(biāo)志GFAP以及神經(jīng)元細(xì)胞的分子標(biāo)志β-tubulin Ⅲ;2.高通量測序的分析結(jié)果顯示共有94個miRNAs在U87細(xì)胞和U87s細(xì)胞間差異表達(dá),其中miR-92a在U87s細(xì)胞中顯著低表達(dá)。qPCR鑒定結(jié)果表明miR-92a在U87s細(xì)胞和U251s細(xì)胞中均顯著下調(diào);3.下調(diào)miR-92a的表達(dá)量在體外可以有效的降低U87細(xì)胞和U251細(xì)胞的增殖和轉(zhuǎn)移能力,同時促進(jìn)細(xì)胞凋亡;4.降低miR-92a的表達(dá)水平可以有效的抑制腦膠質(zhì)瘤裸鼠模型體內(nèi)腫瘤的生長;5.在GSCs細(xì)胞中上調(diào)miR-92a的表達(dá)量后,腫瘤干細(xì)胞球直徑變小、細(xì)胞遷移能力減弱、腫瘤干細(xì)胞球形成率降低,細(xì)胞干性維持相關(guān)分子標(biāo)志表達(dá)量下調(diào);6.生物信息學(xué)預(yù)測和雙熒光素酶報告系統(tǒng)結(jié)果表明miR-92a在腦膠質(zhì)瘤細(xì)胞和GSCs細(xì)胞中可作用于不同的靶基因,miR-92a在腦膠質(zhì)瘤細(xì)胞中的靶基因為CDH1,在GSCs細(xì)胞中的靶基因則為Notch-1;7.腦膠質(zhì)瘤細(xì)胞內(nèi)miR-92a的表達(dá)量下調(diào)后,CDH1蛋白量增多,細(xì)胞核內(nèi)p-catenin含量減少,而總的p-catenin含量不變;8.在GSCs細(xì)胞內(nèi)過表達(dá)miR-92a后,Notch-M蛋白下調(diào),細(xì)胞內(nèi)磷酸化Akt的含量減少,而總的Akt含量不變;9.過表達(dá)CDH1可以抑制腦膠質(zhì)瘤細(xì)胞的轉(zhuǎn)移能力,而降低Notch-1的表達(dá)則可抑制GSCs細(xì)胞的生長、遷移和自我更新能力。結(jié)論本研究首先從腦膠質(zhì)瘤細(xì)胞中分離出GSCs細(xì)胞,通過高通量測序技術(shù)發(fā)現(xiàn)miR-92a在兩種類型的細(xì)胞中差異表達(dá)。qPCR結(jié)果證實,與腦膠質(zhì)瘤細(xì)胞相比,GSCs細(xì)胞內(nèi)miR-92a的含量明顯降低。降低miR-92a的表達(dá)水平可有效抑制腦膠質(zhì)瘤細(xì)胞的增殖、遷移、侵襲,并促進(jìn)細(xì)胞凋亡;miR-92a含量減少后,腦膠質(zhì)瘤裸鼠模型體內(nèi)腫瘤生長速度也明顯減慢。另一方面,上調(diào)miR-92a的含量可以顯著抑制GSCs細(xì)胞的生長,遷移和自我更新。深入的機制研究表明,miR-92a可以通過分別作用于下游CDHl/p-catenin和Notch-1/Akt信號通路,影響腦膠質(zhì)瘤細(xì)胞和GSCs細(xì)胞的惡性表型。上述實驗結(jié)果表明miR-92a在腦膠質(zhì)瘤的形成和發(fā)展過程中起重要作用。
[Abstract]:Glioma is one of the most common primary tumors of the central nervous system with high malignant level and high morbidity and mortality. In recent years, although people have made considerable progress in surgical treatment of glioma, radiotherapy and chemotherapy, the prognosis of patients with malignant glioma is still poor and after diagnosis. The average life cycle is not more than 15 months. The latest research has found a small group of cell subgroups with stem cell characteristics in glioma cells, which can continue to proliferate, have self-renewal ability and multidirectional differentiation potential, and are defined as brain glioma stem cells (Glioma stem cells, GSCs).GSCs cells are brain glue The main cause of tumor formation, infiltration, metastasis, recurrence and chemotherapy tolerance.MicroRNAs (miRNAs) is a class of non coded RNA molecules, which are highly conserved in evolution and are about 22nt around 22nt, and can prevent mRNAs's transcription by directly acting on the 3 terminal non translation region (3'Untranslated Regions, 3'UTR) downstream of the downstream mRNAs, and the domain induces mRNAs. There is a growing number of evidence that miRNAs molecules can play a role similar to proto oncogenes or tumor suppressor genes to affect the formation and development of tumor cells. In addition, there are also reports that miRNAs also plays a role in the regulation of differentiation and self renewal of cancer stem cells. Therefore, it is possible to reduce the malignancy of tumor cells and tumor stem cells by changing the expression of miRNAs, which may be a promising treatment. In this study, the authors found that the level of miR-92a expression in GSCs cells decreased significantly compared with glioma cells. Low. Previous reports suggest that miR-92a is highly expressed in the tissue specimens of glioma cell lines and glioma patients. Reducing the expression level of miR-92a can inhibit the proliferation of glioma cells and promote cell apoptosis. However, miR-92a migration, invasion, and GSCs cell survival, migration and self renewal in glioma cells. The function and specific molecular mechanism of miR-92a in these two cells can provide new ideas for the treatment of glioma. The purpose of this study is to explore the function and detailed mechanism of miR-92a in glioma and GSCs cells, and to provide a new theoretical basis for the targeting therapy of glioma. Methods 1. the GSCs cells were isolated and cultured from glioma cells (U87 and U251) by serum-free starvation, and GSCs cells were identified by immunofluorescence staining. 2. the difference of miRNAs expression in U87 cells and U87 derived GSCs cells U87s was compared with second generation high-throughput sequencing technology; 3. used MTT, cell scratch, Transwell, and An. Nexin V-FITC/PI double staining was used to detect the effect of miR-92a on the proliferation, migration, invasion and apoptosis of U87 and U251 cells in vitro. 4. the effect of miR-92a on the growth of glioma cells in vivo was detected by nude mice, and the growth, migration and self of miR-92a to GSCs cells were detected by qPCR, Western Blot and tumor stem cells. The effect of update; 6. combined with bioinformatics and double luciferase reporting system to predict and identify the target genes of miR-92a in brain glioma cells and GSCs cells; 7. the molecular mechanism of miR-92a in glioma and GSCs cells was determined by Western Blot and immunofluorescence staining. Experimental results 1. use serum-free starvation. GSCs cells (U87s and U251s) were isolated from brain glioma cells (U87 and U251). The results of immunofluorescence staining showed that GSCs cells expressed the molecular markers CD133 of tumor stem cells and molecular markers Nestin of neural stem cells, and the molecular markers of neuroglial cells expressed as GFAP and neuron cells were expressed in the differentiated GSCs cells. The molecular marker beta -tubulin III; 2. high throughput sequencing results showed a total of 94 miRNAs differentially expressed between U87 cells and U87s cells, and the significant low expression of.QPCR identification in U87s cells showed that miR-92a was significantly downregulated in U87s and U251s cells, and 3. down-regulation of miR-92a expression was effective in vitro. Reduce the proliferation and metastasis ability of U87 and U251 cells, and promote cell apoptosis. 4. reducing the expression level of miR-92a can effectively inhibit the growth of tumor in the nude mice model of glioma. 5. after the expression of miR-92a in GSCs cells, the diameter of the tumor stem cells becomes smaller, the cell migration ability is weakened, and the tumor stem cells are spherical. 6. bioinformatics prediction and double luciferase reporter system results showed that miR-92a could play a role in different target genes in brain glioma cells and GSCs cells. The target gene of miR-92a in glioma cells was CDH1, and the target gene in GSCs cells was Notch-1; 7 After the expression of miR-92a in glioma cells was down, the amount of CDH1 protein increased, the content of P-catenin in the nucleus decreased and the total P-catenin content was unchanged. 8. after overexpression of miR-92a in GSCs cells, the Notch-M protein was down, the content of phosphorylated Akt in the cell decreased and the total Akt content was unchanged; 9. overexpression CDH1 could inhibit glioma fine. The expression of Notch-1 can inhibit the growth, migration and self renewal of GSCs cells. Conclusion this study first isolated GSCs cells from brain glioma cells. Through high throughput sequencing, the differential expression of miR-92a in two types of cells was found to be confirmed by.QPCR, compared with glioma cells, GSCs The content of miR-92a in cells decreased significantly. Reducing the expression level of miR-92a could effectively inhibit the proliferation, migration, invasion and apoptosis of glioma cells. After the decrease of miR-92a, the growth rate of tumor in the nude mice model of brain glioma was also slowed down. On the other hand, up regulation of miR-92a could significantly inhibit the GSCs cells. Growth, migration and self renewal. Deep mechanism studies have shown that miR-92a can affect the malignant phenotype of glioma cells and GSCs cells by acting on the downstream CDHl/p-catenin and Notch-1/Akt signaling pathways respectively. These results suggest that miR-92a plays an important role in the formation and development of glioma.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R739.41

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7 金源;中關(guān)村生命科學(xué)園構(gòu)建國家生物醫(yī)藥基地[N];中國醫(yī)藥報;2006年

8 張獻(xiàn)懷;對腦膠質(zhì)瘤應(yīng)進(jìn)行局部強力打擊[N];中國醫(yī)藥報;2007年

9 健康時報特約記者 朱立明;切腦膠質(zhì)瘤用超聲引導(dǎo)[N];健康時報;2007年

10 通訊員 張獻(xiàn)懷;腦膠質(zhì)瘤臨床研究與治療獲新進(jìn)展[N];大眾科技報;2009年

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10 潘俊辰;異常表達(dá)長鏈非編碼RNA在腦膠質(zhì)瘤中的功能研究[D];南京醫(yī)科大學(xué);2015年

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