本文關鍵詞： 微血管內皮細胞增殖 MiR-7-5p 膠質母細胞瘤 RAF1 慢病毒包裝　出處：《山東大學》2014年博士論文　論文類型：學位論文

【摘要】：背景 惡性腫瘤的發(fā)生與發(fā)展是一個極為復雜的過程,多種基因、多種信號通路參與其中,多種相關的發(fā)育基因在此過程中發(fā)揮著異常重要的作用。MicroRNAs的表達與調控作用,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越多的研究。MicroRNAs (miRNAs)是一類長度為18-22nt、具有基因調控功能的小分子非編碼RNA。微RNAs (microRNAs, miRNAs)的失調往往與癌癥的發(fā)生和發(fā)展相關。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結合,引起靶mRNAs的降解,或者抑制其翻譯,從而發(fā)揮其生物學作用。 膠質母細胞瘤(Glioblastoma, WHO IV級),易稱為多形性膠質母細胞瘤(Glioblastoma multiforme, GBM),無論是從發(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,膠質母細胞瘤的治療,一直是神經(jīng)外科學領域的一大難題,目前主要的臨床治療方案仍然是手術中最大限度的切除及輔以術后放療、化療等手段。雖然近幾年來對GBM的有些治療取得了新的進展,但由于其高度惡性,高術后復發(fā)率,預后仍然較差。因此,目前探索新的GBM的發(fā)病機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。 微血管增殖是GBM特有的病理特征,但是,目前對于增殖的GBM微血管內皮細胞的功能知之甚少。幾乎所有的研究與化療藥物均是靶向作用于GBM細胞,而作用于抗腫瘤血管生成的化療藥物未出現(xiàn)預期的效果。GBM中微血管只占到腫瘤很小的一部分,以往研究通過提取整個腫瘤的總RNA或基因組DNA來尋找GBM微血管特異性差異基因顯然是不適當?shù)�。本研究將提取非腫瘤腦組織微血管和GBM微血管,用微芯片技術分析對比GBM微血管和腦組織微血管的miRNA表達譜的差異性,應用現(xiàn)代生物學信息工具分析整合miRNA表達譜,在miRNA、mRNA及蛋白水平上研究能夠加強或抑制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。 目的 1.提取純化GBM及正常腦組織的微血管,檢測二者的差異miRNAs; 2.篩選出差異顯著的miRNA,研究其對血管內皮細胞生物學行為的影響； 3.對篩選出的差異顯著的miRNA進行功能及其機制的研究,為明確其是否可作為GBM治療的新靶點提供實驗依據(jù)。 方法 1.應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。以此為基點提取總RNA。 2.用miRNA基因芯片技術,篩查在GBM微血管和腦組織微血管中的差異表達的microRNAs,應用實時定量PCR進一步對其進行驗證,驗證其在微血管組織及在人臍靜脈血管內皮細胞系(HUV-EC-C)中的表達情況。 3.運用生物信息學工具TagetScan5.1、MicroCosm Targets Version5預測差異miRNA的靶基因,并對其特異性靶基因進行KEGG pathway分析。選擇確定差異顯著的參與調控血管內皮細胞生物學行為的microRNA及其靶基因。 4.構建過表達目的基因的慢病毒載體和陰性對照慢病毒載體；應用293T細胞進行病毒包裝和滴度測定；慢病毒載體感染HUV-EC-C細胞；應用qPCR檢測慢病毒載體目的基因的表達。 5.采用劃痕實驗、MTT實驗、流式細胞術,觀察目的基因的表達對血管內皮細胞體外遷移能力、增殖能力及其細胞周期的影響。 6.將預測的靶基因的野生型或突變型3'-UTR克隆入GV272的XbaI位點構建螢光素酶報告基因,與過表達miRNA慢病毒或對照組慢病毒質粒共轉染HUV-EC-C細胞,通過分析螢光素酶的表達以初步判定miRNA的靶基因。應用Western Blot在細胞及組織中檢測,進一步確認miRNA對其靶基因的調控關系。 7.所有結果用均數(shù)加減標準差來表示。t檢驗進行組間比較。MiR-7-5p和RAF1表達之間的關系通過皮爾森相關性統(tǒng)計方法來探究。P0.05為差異有統(tǒng)計學意義。所有統(tǒng)計學分析用SPSS16.0軟件進行。 結果 1.應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,并應用臺盼蘭染色,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,呈團狀,分支雜亂。 2.通過基因芯片分析顯示,在GBM微血管中miR-7-5p相對于正常腦組織微血管呈現(xiàn)大幅下調(P0.01),相對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,應用qPCR驗證符合基因芯片分析結果。之后,我們又在人臍靜脈內皮細胞系(HUV-EC-C)中驗證miR-7-5p的表達情況,經(jīng)qPCR檢測,HUV-EC-C細胞中miR-7-5p明顯低表達。 3.生物信息學預測結果表明,參與血管內皮細胞生物學行為的RAF1的3'-UTR與miR-7-5p的5’端“種子序列”有很好的匹配位點。熒光素酶活性測定結果顯示RAF1是miR-7-5p下游的直接靶點(P0.01)。 4.MTT實驗、細胞周期實驗顯示,在體外miR-7-5p誘導血管內皮細胞周期停滯于G1期,從而抑制血管內皮細胞增值。劃痕實驗表明在體外miR-7-5p對血管內皮細胞的遷移能力無影響。 5. Western blot分析顯示,在HUV-EC-C細胞感染過表達miR-7-5p病毒能降低RAF1的蛋白水平,Actin作為內參,(P0.01)；在GBM和正常腦組織微血管中,miR-7-5p和RAF1的表達呈負相關關系。Pearson's相關分析具有統(tǒng)計學‘意義。相關性系數(shù)為-0.786,(P0.01)。 結論 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開始。因此,對于明確在GBM微血管的發(fā)生發(fā)展中起至關重要的有意義的microRNAs及其它們的作用靶點,具有更好的參考價值,對GBM的治療可能提供更有意義的實驗依據(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,并應用qPCR在GBM微血管組織及HUVEC-C細胞株中進行了驗證。此外,我們成功地包裝了過表達miR-7-5p及其對照組慢病毒,并進行了體外MTT實驗、細胞周期實驗及劃痕實驗。實驗結果表明niR-7-5p的過度表達能夠通過誘導HUV-EC-C細胞在G1期的細胞周期停滯而顯著抑制其增殖,而對血管內皮細胞在體外的遷移能力無影響。這些結果有力地支持了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介導的GBM微血管內皮細胞生長受抑制的機制,我們接下來開始鑒別miR-7-5p潛在的靶基因。生物信息學分析顯示,RAF1可能是]miR-7-5p潛在的下游靶點。而且,RAF1在多種癌癥的發(fā)生發(fā)展中起著重要作用。因此,我們應用熒光素酶活性測定法來鑒定miR-7-5p在RAF1表達上的作用。熒光素酶活性測定數(shù)據(jù)顯示miR-7-5p能夠直接靶向作用于RAF1的3'-UTR。蛋白免疫印跡結果顯示,在HUV-EC-C細胞中miR-7-5p的過度表達明顯下調了RAF1的表達。我們又對5組GBM微血管和正常腦組織微血管進行了western blot實驗,結果顯示RAF1在GBM微血管組織中上調,在腦組織微血管中下調,并且和miR-7-5p的表達呈負相關關系。這些數(shù)據(jù)證實了miR-7-5p能夠下調RAF1的表達,這也表明了RAF1在GBM微血管的形成中可能是一個促進因素。本研究提示,miR-7-5p通過對下游靶基因RAF1的調控,在GBM微血管的形成和發(fā)展中可能扮演抑癌基因的角色,并在調控血管內皮細胞的增殖等惡性生物學特性中發(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 .

【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R739.41

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