本文關(guān)鍵詞： 微血管內(nèi)皮細(xì)胞增殖 MiR-7-5p 膠質(zhì)母細(xì)胞瘤 RAF1 慢病毒包裝　出處：《山東大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：背景 惡性腫瘤的發(fā)生與發(fā)展是一個(gè)極為復(fù)雜的過(guò)程,多種基因、多種信號(hào)通路參與其中,多種相關(guān)的發(fā)育基因在此過(guò)程中發(fā)揮著異常重要的作用。MicroRNAs的表達(dá)與調(diào)控作用,作為腫瘤發(fā)生發(fā)展的重要因素之一,被日益受到重視和越來(lái)越多的研究。MicroRNAs (miRNAs)是一類長(zhǎng)度為18-22nt、具有基因調(diào)控功能的小分子非編碼RNA。微RNAs (microRNAs, miRNAs)的失調(diào)往往與癌癥的發(fā)生和發(fā)展相關(guān)。miRNAs靶向作用于其靶基因mRNAs,并與其3'-UTR完全或者不完全互補(bǔ)結(jié)合,引起靶mRNAs的降解,或者抑制其翻譯,從而發(fā)揮其生物學(xué)作用。 膠質(zhì)母細(xì)胞瘤(Glioblastoma, WHO IV級(jí)),易稱為多形性膠質(zhì)母細(xì)胞瘤(Glioblastoma multiforme, GBM),無(wú)論是從發(fā)病率還是惡性程度,都是中樞神經(jīng)系統(tǒng)腫瘤中最高的原發(fā)性腫瘤。多年來(lái),膠質(zhì)母細(xì)胞瘤的治療,一直是神經(jīng)外科學(xué)領(lǐng)域的一大難題,目前主要的臨床治療方案仍然是手術(shù)中最大限度的切除及輔以術(shù)后放療、化療等手段。雖然近幾年來(lái)對(duì)GBM的有些治療取得了新的進(jìn)展,但由于其高度惡性,高術(shù)后復(fù)發(fā)率,預(yù)后仍然較差。因此,目前探索新的GBM的發(fā)病機(jī)制及尋找其新的更為有效的治療方法仍然是一大研究熱點(diǎn)。 微血管增殖是GBM特有的病理特征,但是,目前對(duì)于增殖的GBM微血管內(nèi)皮細(xì)胞的功能知之甚少。幾乎所有的研究與化療藥物均是靶向作用于GBM細(xì)胞,而作用于抗腫瘤血管生成的化療藥物未出現(xiàn)預(yù)期的效果。GBM中微血管只占到腫瘤很小的一部分,以往研究通過(guò)提取整個(gè)腫瘤的總RNA或基因組DNA來(lái)尋找GBM微血管特異性差異基因顯然是不適當(dāng)?shù)�。本研究將提取非腫瘤腦組織微血管和GBM微血管,用微芯片技術(shù)分析對(duì)比GBM微血管和腦組織微血管的miRNA表達(dá)譜的差異性,應(yīng)用現(xiàn)代生物學(xué)信息工具分析整合miRNA表達(dá)譜,在miRNA、mRNA及蛋白水平上研究能夠加強(qiáng)或抑制GBM微血管增殖的某些因子,以期發(fā)現(xiàn)更有效的潛在抑制GBM微血管增殖的新靶點(diǎn)。 目的 1.提取純化GBM及正常腦組織的微血管,檢測(cè)二者的差異miRNAs; 2.篩選出差異顯著的miRNA,研究其對(duì)血管內(nèi)皮細(xì)胞生物學(xué)行為的影響； 3.對(duì)篩選出的差異顯著的miRNA進(jìn)行功能及其機(jī)制的研究,為明確其是否可作為GBM治療的新靶點(diǎn)提供實(shí)驗(yàn)依據(jù)。 方法 1.應(yīng)用葡聚糖沉淀梯度密度離心法,提取純化GBM微血管和非腫瘤正常腦組織微血管。以此為基點(diǎn)提取總RNA。 2.用miRNA基因芯片技術(shù),篩查在GBM微血管和腦組織微血管中的差異表達(dá)的microRNAs,應(yīng)用實(shí)時(shí)定量PCR進(jìn)一步對(duì)其進(jìn)行驗(yàn)證,驗(yàn)證其在微血管組織及在人臍靜脈血管內(nèi)皮細(xì)胞系(HUV-EC-C)中的表達(dá)情況。 3.運(yùn)用生物信息學(xué)工具TagetScan5.1、MicroCosm Targets Version5預(yù)測(cè)差異miRNA的靶基因,并對(duì)其特異性靶基因進(jìn)行KEGG pathway分析。選擇確定差異顯著的參與調(diào)控血管內(nèi)皮細(xì)胞生物學(xué)行為的microRNA及其靶基因。 4.構(gòu)建過(guò)表達(dá)目的基因的慢病毒載體和陰性對(duì)照慢病毒載體；應(yīng)用293T細(xì)胞進(jìn)行病毒包裝和滴度測(cè)定；慢病毒載體感染HUV-EC-C細(xì)胞；應(yīng)用qPCR檢測(cè)慢病毒載體目的基因的表達(dá)。 5.采用劃痕實(shí)驗(yàn)、MTT實(shí)驗(yàn)、流式細(xì)胞術(shù),觀察目的基因的表達(dá)對(duì)血管內(nèi)皮細(xì)胞體外遷移能力、增殖能力及其細(xì)胞周期的影響。 6.將預(yù)測(cè)的靶基因的野生型或突變型3'-UTR克隆入GV272的XbaI位點(diǎn)構(gòu)建螢光素酶報(bào)告基因,與過(guò)表達(dá)miRNA慢病毒或?qū)φ战M慢病毒質(zhì)粒共轉(zhuǎn)染HUV-EC-C細(xì)胞,通過(guò)分析螢光素酶的表達(dá)以初步判定miRNA的靶基因。應(yīng)用Western Blot在細(xì)胞及組織中檢測(cè),進(jìn)一步確認(rèn)miRNA對(duì)其靶基因的調(diào)控關(guān)系。 7.所有結(jié)果用均數(shù)加減標(biāo)準(zhǔn)差來(lái)表示。t檢驗(yàn)進(jìn)行組間比較。MiR-7-5p和RAF1表達(dá)之間的關(guān)系通過(guò)皮爾森相關(guān)性統(tǒng)計(jì)方法來(lái)探究。P0.05為差異有統(tǒng)計(jì)學(xué)意義。所有統(tǒng)計(jì)學(xué)分析用SPSS16.0軟件進(jìn)行。 結(jié)果 1.應(yīng)用葡聚糖沉淀梯度密度離心法從GBM和正常腦組織中成功純化提取出微血管組織,并應(yīng)用臺(tái)盼蘭染色,鏡下可見正常腦組織微血管每條清晰完整,分支明顯；GBM微血管破碎,呈團(tuán)狀,分支雜亂。 2.通過(guò)基因芯片分析顯示,在GBM微血管中miR-7-5p相對(duì)于正常腦組織微血管呈現(xiàn)大幅下調(diào)(P0.01),相對(duì)于正常非腫瘤腦組織微血管減少2.616倍。在各5例GBM微血管和腦組織微血管中,應(yīng)用qPCR驗(yàn)證符合基因芯片分析結(jié)果。之后,我們又在人臍靜脈內(nèi)皮細(xì)胞系(HUV-EC-C)中驗(yàn)證miR-7-5p的表達(dá)情況,經(jīng)qPCR檢測(cè),HUV-EC-C細(xì)胞中miR-7-5p明顯低表達(dá)。 3.生物信息學(xué)預(yù)測(cè)結(jié)果表明,參與血管內(nèi)皮細(xì)胞生物學(xué)行為的RAF1的3'-UTR與miR-7-5p的5’端“種子序列”有很好的匹配位點(diǎn)。熒光素酶活性測(cè)定結(jié)果顯示RAF1是miR-7-5p下游的直接靶點(diǎn)(P0.01)。 4.MTT實(shí)驗(yàn)、細(xì)胞周期實(shí)驗(yàn)顯示,在體外miR-7-5p誘導(dǎo)血管內(nèi)皮細(xì)胞周期停滯于G1期,從而抑制血管內(nèi)皮細(xì)胞增值。劃痕實(shí)驗(yàn)表明在體外miR-7-5p對(duì)血管內(nèi)皮細(xì)胞的遷移能力無(wú)影響。 5. Western blot分析顯示,在HUV-EC-C細(xì)胞感染過(guò)表達(dá)miR-7-5p病毒能降低RAF1的蛋白水平,Actin作為內(nèi)參,(P0.01)；在GBM和正常腦組織微血管中,miR-7-5p和RAF1的表達(dá)呈負(fù)相關(guān)關(guān)系。Pearson's相關(guān)分析具有統(tǒng)計(jì)學(xué)‘意義。相關(guān)性系數(shù)為-0.786,(P0.01)。 結(jié)論 葡聚糖沉淀梯度密度離心法能夠成功提取GBM微血管與正常腦組織微血管,臺(tái)盼蘭染色,鏡下發(fā)現(xiàn)二者在形態(tài)上有明顯的差別,這符合客觀規(guī)律。本研究從純化的GBM微血管和正常腦組織的微血管為基點(diǎn)開始。因此,對(duì)于明確在GBM微血管的發(fā)生發(fā)展中起至關(guān)重要的有意義的microRNAs及其它們的作用靶點(diǎn),具有更好的參考價(jià)值,對(duì)GBM的治療可能提供更有意義的實(shí)驗(yàn)依據(jù)。在目前基因芯片研究中,我們揭示了miR-7-5p在GBM微血管中明顯下調(diào),并應(yīng)用qPCR在GBM微血管組織及HUVEC-C細(xì)胞株中進(jìn)行了驗(yàn)證。此外,我們成功地包裝了過(guò)表達(dá)miR-7-5p及其對(duì)照組慢病毒,并進(jìn)行了體外MTT實(shí)驗(yàn)、細(xì)胞周期實(shí)驗(yàn)及劃痕實(shí)驗(yàn)。實(shí)驗(yàn)結(jié)果表明niR-7-5p的過(guò)度表達(dá)能夠通過(guò)誘導(dǎo)HUV-EC-C細(xì)胞在G1期的細(xì)胞周期停滯而顯著抑制其增殖,而對(duì)血管內(nèi)皮細(xì)胞在體外的遷移能力無(wú)影響。這些結(jié)果有力地支持了niR-7-5p可能是GBM微血管增值的一種抑制物。 為了探究引起由miR-7-5p介導(dǎo)的GBM微血管內(nèi)皮細(xì)胞生長(zhǎng)受抑制的機(jī)制,我們接下來(lái)開始鑒別miR-7-5p潛在的靶基因。生物信息學(xué)分析顯示,RAF1可能是]miR-7-5p潛在的下游靶點(diǎn)。而且,RAF1在多種癌癥的發(fā)生發(fā)展中起著重要作用。因此,我們應(yīng)用熒光素酶活性測(cè)定法來(lái)鑒定miR-7-5p在RAF1表達(dá)上的作用。熒光素酶活性測(cè)定數(shù)據(jù)顯示miR-7-5p能夠直接靶向作用于RAF1的3'-UTR。蛋白免疫印跡結(jié)果顯示,在HUV-EC-C細(xì)胞中miR-7-5p的過(guò)度表達(dá)明顯下調(diào)了RAF1的表達(dá)。我們又對(duì)5組GBM微血管和正常腦組織微血管進(jìn)行了western blot實(shí)驗(yàn),結(jié)果顯示RAF1在GBM微血管組織中上調(diào),在腦組織微血管中下調(diào),并且和miR-7-5p的表達(dá)呈負(fù)相關(guān)關(guān)系。這些數(shù)據(jù)證實(shí)了miR-7-5p能夠下調(diào)RAF1的表達(dá),這也表明了RAF1在GBM微血管的形成中可能是一個(gè)促進(jìn)因素。本研究提示,miR-7-5p通過(guò)對(duì)下游靶基因RAF1的調(diào)控,在GBM微血管的形成和發(fā)展中可能扮演抑癌基因的角色,并在調(diào)控血管內(nèi)皮細(xì)胞的增殖等惡性生物學(xué)特性中發(fā)揮著重要作用。
[Abstract]:Background MicroRNAs ( microRNAs ) are a kind of small molecule non - coding RNAs with a length of 18 - 22 nt and have gene regulation function . MicroRNAs ( microRNAs ) are a kind of small molecule non - coding RNAs with a length of 18 - 22nt and have gene regulation function . MicroRNAs ( microRNAs ) are a kind of small molecule non - coding RNAs with length of 18 - 22nt . Glioblastoma ( WHO grade IV ) , known as Glioblastoma multiforme , is one of the most important primary tumors in the central nervous system tumor . Microchip technique is used to study the difference of miRNA expression profiles of microvessels and brain tissues of non - tumor tissues . Purpose ( 1 ) extracting and purifying the microvessels of the purified and normal brain tissues , and detecting the difference miRNA of the two ; 2 . screening the miRNA with obvious difference to study the effect of the miRNA on the biological behavior of vascular endothelial cells ; 3 . The study of the function and mechanism of miRNA with significant difference in screening can provide an experimental basis for determining whether it can be used as a new target for the treatment . method 1 . Using dextran precipitation gradient density centrifugation method , the microvessels of the microvessels and non - tumor normal brain tissues were purified and purified . The total RNA was extracted by using the method as the base point . 2 . Using miRNA gene chip technique to screen microRNAs expressed differentially expressed in microvessels and brain tissue , real - time quantitative PCR was used to further verify the expression of microRNAs in microvascular tissue and human umbilical vein endothelial cell line ( HUV - EC - C ) . 3 . Using the bioinformatic tool TagetScan5.1 , MicroCosm Targets Version 5 , the target gene of the difference miRNA was predicted , and its specific target gene was analyzed . The microRNAs and their target genes involved in regulating the biological behavior of vascular endothelial cells were selected . 4 . The lentivirus vector and the negative control slow virus vector expressing the target gene were constructed ; the virus packaging and the titer determination were carried out by the expression target gene ; the lentivirus vector was infected with HUV - EC - C cells ; and the expression of the target gene of the lentivirus vector was detected by qPCR . 5 . MTT assay and flow cytometry were used to observe the effect of gene expression on migration , proliferation and cell cycle of vascular endothelial cells in vitro . 6 . The luciferase reporter gene was constructed by cloning the wild - type or mutant 3 ' - terminal of the predicted target gene into the Xba I site of GV272 . HUV - EC - C cells were co - transfected with a slow virus plasmid expressing the miRNA lentivirus or the control group . The expression of luciferase was analyzed to determine the target gene of miRNA . Western Blot was used to detect the target gene in the cells and tissues , and the regulatory relationship between miRNA and its target gene was further confirmed . 7 . All results were expressed by mean addition and subtraction standard deviation . The relationship between MiR - 7 - 5p and RAF1 expression was investigated by Pearson correlation statistical method . All statistical analyses were performed by SPSS 16.0 software . Results 1 . Microvascular tissue was successfully purified by dextran precipitation gradient density centrifugation and trypan blue staining was used in normal brain tissue . 2 . The expression of miR - 7 - 5p in human umbilical vein endothelial cell line ( HUV - EC - C ) was significantly downregulated ( P0.01 ) , and the expression of miR - 7 - 5p in human umbilical vein endothelial cell line ( HUV - EC - C ) was verified by qPCR , and the expression of miR - 7 - 5p in HUV - EC - C cells was significantly lower than that in normal non - tumor tissue . 3 . The results of bioinformatics predict that the 3 ' - untranslated region of RAF1 participating in the biological behavior of vascular endothelial cells has a good matching site with the 5 ' - terminal " seed sequence " of miR - 7 - 5p . The luciferase activity assay results show that RAF1 is a direct target downstream of miR - 7 - 5p ( P0.01 ) . 4 . MTT assay and cell cycle experiment show that miR - 7 - 5p induces vascular endothelial cell cycle arrest in G1 phase in vitro , thereby inhibiting the value of vascular endothelial cells . The scratch test shows that miR - 7 - 5p has no effect on the migration ability of vascular endothelial cells in vitro . 5 . Western blot analysis showed that the expression of miR - 7 - 5p in HUV - EC - C cells decreased RAF1 protein level and Actin as internal reference , ( P0.01 ) . Conclusion The results show that the overexpression of niR - 7 - 5p can significantly inhibit the proliferation of vascular endothelial cells and the migration ability of vascular endothelial cells in vitro . These results strongly support niR - 7 - 5p , which may be a kind of inhibitor of the value - added of the microvascular endothelial cells . In order to investigate the mechanism of miR - 7 - 5p mediated inhibition of expression of miR - 7 - 5p , we began to identify the potential target genes of miR - 7 - 5p .

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.41

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本文編號(hào)：1529044