本文選題：膠質(zhì)瘤 + 外泌體��； 參考：《河北醫(yī)科大學》2017年博士論文

【摘要】：腦膠質(zhì)瘤在中樞神經(jīng)系統(tǒng)中高發(fā),且預后很差。即使給予術(shù)后放療、化療或聯(lián)合治療,腦膠質(zhì)瘤患者的復發(fā)率仍然很高,治療效果始終不能令人滿意。隨著基因水平研究的不斷深入,有關(guān)膠質(zhì)瘤的基因分子機制得到深入的探索。O6-甲基鳥嘌呤-DNA甲基轉(zhuǎn)移酶(O6-methylguanine-DNA methyhransferase,MGMT)啟動子甲基化、1p/19q雜合性缺失、異檸檬酸脫氫酶1(Isocitrate dehydrogenase 1,IDH1)基因突變、ATRX表達缺失等均已經(jīng)成為膠質(zhì)瘤病理學診斷的重要分子生物學標記,也豐富了人們對于腫瘤基因變異的認識。Stephen Paget于1889年提出腫瘤生長的“種子-土壤”學說,奠定了腫瘤微環(huán)境的概念。外泌體是腫瘤微環(huán)境中的重要成分。它通過釋放自身攜帶的細胞因子參與了腫瘤的發(fā)生、增殖、侵襲和化療抵抗等生物學過程。而腫瘤細胞來源的外泌體(Tumor cells derived exosome,Texo)由于它的結(jié)構(gòu)特征和運輸功能已經(jīng)成為近年來腫瘤領(lǐng)域的研究熱點。2013年,美國、德國3位科學家憑借他們所發(fā)現(xiàn)的細胞囊泡運輸?shù)恼{(diào)節(jié)機制,榮獲2013年諾貝爾生理學或醫(yī)學獎。外泌體(exosome)作為人體內(nèi)一類重要運輸囊泡,也開始受到越來越多的關(guān)注�？茖W家們已發(fā)現(xiàn),外泌體會參與到腫瘤細胞的生長、增殖、侵襲、凋亡、血管生成、炎癥反應、免疫應答等重要的生物過程,細胞會通過分泌外泌體,將一些信號分子分泌到較遠的組織或細胞中,以起到調(diào)控作用。外泌體作為藥物傳遞系統(tǒng)(Drug delivery system,DDS)為高效藥物投遞提供天然的內(nèi)源性納米級載體,其靶向作用的潛力也逐漸被發(fā)現(xiàn)。miRNA是一類內(nèi)源性的具有調(diào)控功能的非編碼RNA,可以與靶mRNA的3'非翻譯區(qū)(Untranslated Regions,UTR)結(jié)合從而在轉(zhuǎn)錄后水平調(diào)節(jié)靶基因的表達。有研究顯示,miR-221在直腸癌、胰腺癌、乳腺癌和膀胱癌中是一種促癌基因。利用高通量篩選測序平臺篩選腦膠質(zhì)瘤組織miRNA差異表達情況,篩選出了膠質(zhì)瘤組織內(nèi)miR-221高表達,特別是膠質(zhì)母細胞瘤組織�？紤]到膠質(zhì)瘤細胞外泌體內(nèi)miR-221的表達情況以及調(diào)節(jié)機制尚未進行過研究,故在本實驗中我們選取膠質(zhì)瘤細胞來源的外泌體miR-221作為研究對象,進行體內(nèi)外細胞和外泌體miR-221表達的檢測,并通過CCK-8實驗,流式細胞技術(shù)以及Transwell實驗探討其對于膠質(zhì)瘤的作用。然后進一步尋找miRNA的靶基因。DNM3(Dynamin 3)是發(fā)動蛋白超家族(包括DNM1、DNM2、DNM3)中的一員,參與許多膜轉(zhuǎn)運功能,例如胞質(zhì)分裂、吞噬作用、轉(zhuǎn)運囊泡出芽和細胞流動等。最近的研究表明,DNM3的高表達可抑制細胞增殖及誘導凋亡�；趯δ壳耙延形墨I的充分分析,我們推測miRNA-DNM3途徑有可能成為抑制神經(jīng)膠質(zhì)瘤生長及放化療增敏的治療靶點。由于DNM3出現(xiàn)在Targetscan的預測結(jié)果中,我們進一步研究其與miR-221的關(guān)系。實驗中我們確定了miR-221的靶基因DNM3以及miR-221的轉(zhuǎn)錄調(diào)控因子RELA,并通過雙熒光素酶報告基因?qū)嶒灥靡宰C實,并分析它們表達量的相關(guān)性,最后通過病毒轉(zhuǎn)染實驗證實miR-221對膠質(zhì)瘤的作用是通過靶向調(diào)控DNM3引起的,并受上游RELA的轉(zhuǎn)錄調(diào)控。第一部分miR-221在人腦膠質(zhì)瘤組織和GBM源性外泌體內(nèi)的表達及其與膠質(zhì)瘤WHO病理分級的關(guān)系目的:研究miR-221在人腦膠質(zhì)瘤組織和GBM源性外泌體內(nèi)的表達,并分析miR-221的表達與膠質(zhì)瘤WHO病理分級的關(guān)系。方法:1 48例膠質(zhì)瘤患者及11例非腫瘤患者組織標本均于2015年采集自河北醫(yī)科大學第二醫(yī)院神經(jīng)外科。包括23名女性,36名男性,年齡從30至68歲。標本采集后迅速放至液氮中冷凍,然后-80℃低溫保存。診斷結(jié)果均由2位病理醫(yī)師根據(jù)WHO分級指導得出。2抽取上述患者的靜脈血,室溫靜置后獲得血清或者采集后立即離心獲得血漿上清液,提取外泌體。3 SHG-44、U87MG、HEB和U251的培養(yǎng),及細胞外泌體的提取。4定量反轉(zhuǎn)錄聚合酶鏈反應(qRT-PCR)方法測定神經(jīng)膠質(zhì)瘤組織或外泌體內(nèi)miR-221含量。5應用t檢驗分析神經(jīng)膠質(zhì)瘤中miR-221含量與膠質(zhì)瘤級別或不同細胞系之間的關(guān)系。結(jié)果:1 miRNA-221在人腦膠質(zhì)瘤組織中表達上調(diào):與對照組織比較,膠質(zhì)瘤組織中miRNA-221均呈高表達(P0.01),其中高度惡性膠質(zhì)瘤組織中miRNA-221表達量均明顯高于低度惡性者(P0.01)。在WHO III和WHO IV膠質(zhì)瘤中表達量較WHO II或非腫瘤組織明顯增高(P0.01)。miR-221的表達與膠質(zhì)瘤的病理分級(WHO分級)明顯相關(guān)。2比較在無細胞的血清和血漿外泌體內(nèi)miR-221的差異表達,可見膠質(zhì)瘤患者與非腫瘤患者miR-221表達量具有顯著性差異(P0.01)。3血漿中的檢測結(jié)果與組織檢測結(jié)果相似:膠質(zhì)瘤患者(n=48)與非腫瘤患者(n=11)相比,血漿外泌體miR-221表達量具有顯著性差異(P0.01)。膠質(zhì)瘤患者血漿外泌體miR-221表達量隨膠質(zhì)瘤病理級別升高表達量顯著升高。4 miR-221在四種膠質(zhì)瘤細胞系中均高表達,且miR-221在U87MG細胞來源的外泌體中表達最高,U251、SHG-44、HEB細胞外泌體內(nèi)表達依次降低。結(jié)論:1 miR-221在人腦膠質(zhì)瘤組織中高表達,在血漿來源或膠質(zhì)瘤細胞系來源的外泌體內(nèi)也存在高表達,且其表達量隨著膠質(zhì)瘤WHO病理分級呈遞增趨勢。高級別(III和IV級)膠質(zhì)瘤miR-221的表達量明顯高于低級別(II級)膠質(zhì)瘤,差別具有統(tǒng)計學意義。2 miR-221的表達量與膠質(zhì)瘤WHO病理分級呈顯著的正相關(guān),預示外泌體miR-221可能用于膠質(zhì)瘤相關(guān)的臨床檢測。第二部分U87MG細胞源性的外泌體miR-221對SHG-44細胞生物學活性的影響目的:研究U87MG細胞源性外泌體miR-221對SHG-44細胞的增殖、侵襲、化療抵抗等方面的影響。方法:1培養(yǎng)U87MG和SHG-44細胞系,提取U87MG細胞系外泌體。2向SHG-44細胞系轉(zhuǎn)染anti-miR-221(miR-221 ASO)以降低細胞內(nèi)miR-221的表達,轉(zhuǎn)染anti-miR-NC(Ctrl ASO)作對照。實驗組SHG-44細胞培養(yǎng)基中同時加入外泌體。3進行CCK-8實驗研究降低miR-221表達后膠質(zhì)瘤細胞增殖能力的變化。4進行流式細胞實驗研究降低miR-221表達對膠質(zhì)瘤細胞凋亡能力和替莫唑胺耐藥的變化。5進行Transwell實驗和劃痕實驗研究降低miR-221表達后膠質(zhì)瘤細胞侵襲能力的變化。結(jié)果:1 qRT-PCR結(jié)果顯示,轉(zhuǎn)染anti-miR-221后,SHG-44細胞內(nèi)miR-221的表達量顯著降低(P0.01)。2 CCK-8實驗結(jié)果顯示,anti-miR-NC+Exo組的增殖活力最高,anti-miR-221組的增殖活力最低,即敲低miR-221的表達后,膠質(zhì)瘤細胞SHG-44的增殖能力顯著降低(P0.01)。3流式細胞實驗結(jié)果顯示,敲低miR-221表達后,SHG-44細胞凋亡能力無明顯改變,但是對替莫唑胺的敏感性顯著增高(P0.01)。4 Transwell實驗和劃痕實驗結(jié)果顯示,敲低miR-221表達后,SHG-44細胞侵襲能力顯著降低(P0.01)。結(jié)論:敲低miR-221表達,可以使SHG-44細胞增殖及侵襲能力顯著降低,凋亡能力雖無改變,但是對替莫唑胺的敏感性增高。miR-221可以影響膠質(zhì)瘤細胞的增殖、侵襲及對替莫唑胺的化療耐藥。第三部分miR-221對下游靶基因DNM3的調(diào)控以及RELA對miR-221轉(zhuǎn)錄能力的影響目的:尋找miR-221的下游作用靶點以及miR-221的轉(zhuǎn)錄調(diào)控因子,深入了解miR-221對膠質(zhì)瘤生物學影響的作用機制。方法:1使用Targetscan數(shù)據(jù)庫預測miR-221的下游作用靶點,并搜集相關(guān)文獻篩選有意義結(jié)果。2構(gòu)建miR-221過表達慢病毒質(zhì)粒,建立miR-221過表達穩(wěn)定SHG-44細胞系,進行穩(wěn)定細胞系的篩選。3雙熒光素酶報告基因檢測用于驗證miR-221與DNM3的作用關(guān)系。4應用qRT-PCR及蛋白質(zhì)印跡(Western blot)方法檢測miR-221與DNM3在SHG-44細胞中的表達量,并分析表達量的相關(guān)性。5通過CCK-8實驗、流式細胞術(shù)、Transwell遷移和侵襲實驗等方法,研究上調(diào)DNM3對膠質(zhì)瘤SHG-44細胞增殖、侵襲和凋亡等生物學行為的影響。6通過生物信息學方法,預測miR-221啟動子區(qū)域的轉(zhuǎn)錄因子結(jié)合位點;利用熒光素酶報告實驗,在SHG-44細胞中驗證此轉(zhuǎn)錄因子與miR-221啟動子的結(jié)合情況;通過上調(diào)或下調(diào)這一轉(zhuǎn)錄因子,采用實時定量PCR方法檢測miR-221的表達變化。結(jié)果:1根據(jù)Targetscan的預測結(jié)果,并結(jié)合相關(guān)文獻資料,發(fā)現(xiàn)DNM3上存在兩個與miR-221的作用靶點。2成功構(gòu)建了miR-221過表達慢病毒載體和穩(wěn)轉(zhuǎn)膠質(zhì)瘤細胞系,miR-221的表達水平顯著升高(P0.01),是對照組的45.5倍。3雙熒光素酶報告檢測結(jié)果表明,相對于miR-NC,miR-221可以顯著抑制熒光素酶的活性(P0.01),而在突變靶位點后此現(xiàn)象消失(P0.01)。4 qRT-PCR檢測膠質(zhì)瘤組織中的DNM3表達量:DNM3表達量隨膠質(zhì)瘤病理級別升高表達量反而降低。Spearman秩相關(guān)分析表明,膠質(zhì)瘤細胞DNM3與miR-221的表達量呈負相關(guān)(Spearman r=-0.908,P0.01)。5轉(zhuǎn)染miR-221過表達載體后,在高表達miR-221的細胞中,DNM3低表達;CCK-8實驗提示,增加DNM3的表達后,細胞增殖能力顯著降低。流式細胞技術(shù)檢測發(fā)現(xiàn),加入替莫唑胺后,DNM3促進了膠質(zhì)瘤細胞的早期凋亡(P0.01)。Transwell實驗和劃痕實驗結(jié)果表明,轉(zhuǎn)染DNM3能夠抑制膠質(zhì)瘤細胞的遷移和侵襲能力。6通過生物信息學方法,預測到miR-221編碼基因的啟動子區(qū)域存在RELA的結(jié)合位點;雙熒光素酶報告基因檢測證實RELA能夠與miR-221基因的啟動子區(qū)域相結(jié)合;實時定量PCR結(jié)果表明,轉(zhuǎn)染RELA后,能誘導miR-221的過表達。結(jié)論:DNM3的mRNA中存在miR-221在膠質(zhì)瘤中的靶向作用位點,轉(zhuǎn)錄因子RELA調(diào)節(jié)miR-221基因的轉(zhuǎn)錄。第四部分miR-221促進SHG-44人腦膠質(zhì)瘤細胞系生長的體內(nèi)研究目的:動物體內(nèi)證實miR-221對膠質(zhì)瘤生物學活性影響。方法:1穩(wěn)定轉(zhuǎn)染miR-221的SHG-44膠質(zhì)瘤細胞系,進行穩(wěn)定細胞系的篩選,對照組轉(zhuǎn)染miR-NC。2應用反轉(zhuǎn)錄定量聚合酶鏈反應(qRT-PCR)方法檢測miR-221在SHG-44細胞中的表達量。3裸小鼠皮下人腦膠質(zhì)瘤模型的建立。4腫瘤生長情況觀察:每隔4天用游標卡尺測量一次腫瘤的長徑(a)及寬徑(b),計算腫瘤體積。結(jié)果:1成功構(gòu)建miR-221過表達的SHG-44膠質(zhì)瘤細胞系,qRT-PCR檢測穩(wěn)定細胞系內(nèi)miR-221的相對表達量為47±1.45,明顯高于Vector轉(zhuǎn)染細胞系(P0.01)。2 miR-221過表達腦膠質(zhì)瘤模型證實腫瘤生長速度明顯增快。帶瘤生存20天后取下腫瘤組織,發(fā)現(xiàn)miR-221過表達組腫瘤重量為對照組腫瘤重量的2倍余(P0.05)。結(jié)論:miR-221過表達促進膠質(zhì)瘤細胞體內(nèi)生長,miR-221是一種促癌因子。
[Abstract]:The brain glioma is high in the central nervous system and has poor prognosis. The recurrence rate of the patients with glioma is still high and the therapeutic effect is still not satisfactory even after the postoperative radiotherapy, chemotherapy or combined therapy. The molecular mechanism of glioma has been deeply explored with the molecular mechanism of the gene level..O6- methyl guanine is deeply explored. Methotrexate -DNA methyltransferase (O6-methylguanine-DNA methyhransferase, MGMT) promoter methylation, 1p/19q heterozygosity deletion, ISO citrate dehydrogenase 1 (Isocitrate dehydrogenase 1, IDH1) gene mutation and ATRX expression loss have become important molecular biomarkers for pathological diagnosis of glioma, and also enrich people's tumor base. .Stephen Paget proposed the "seed soil" theory of tumor growth in 1889, which established the concept of tumor microenvironment. Exocrine is an important component of tumor microenvironment. It participates in the biological processes of tumor occurrence, proliferation, invasion and chemotherapeutic resistance by releasing its own cytokine, and tumor cells. Tumor cells derived exosome (Texo), because of its structural characteristics and transport function, has become a research hotspot in the field of cancer in recent years.2013. In the United States, 3 scientists in Germany were awarded the 2013 Nobel prize for physiology or medicine for their discovery of cellular vesicle transport. The exocrine (exosome) was awarded. As an important type of transport vesicles in the human body, more and more attention has been paid. Scientists have found that exocrine experience is involved in the biological processes such as growth, proliferation, invasion, apoptosis, angiogenesis, inflammatory response, immune response and other important biological processes, and the cells secrete some signal molecules into more distant tissues. Drug delivery system (DDS) provides natural endogenous nanoscale carriers for high efficiency drug delivery. The potential of its targeting is also gradually found to be a kind of endogenous non coded RNA with regulatory function, which can be used with the 3'non translation region of the target mRNA (Untrans). Lated Regions, UTR) combined to regulate the expression of target genes at post transcriptional levels. Studies have shown that miR-221 is a oncogene in rectal cancer, pancreatic cancer, breast and bladder cancer. High throughput screening sequencing platform is used to screen the differential expression of miRNA in brain glioma tissue, and the high expression of miR-221 in glioma tissues is screened, especially in glioma tissues. It is a glioblastoma tissue. Considering the expression of miR-221 in the extracellular secretory of glioma cells and the regulation mechanism has not been studied, in this experiment, we selected the external secretory miR-221 derived from glioma cells as the research object to detect the expression of miR-221 in the cells and exosecrete in vitro and in vivo, and through the CCK-8 experiment, flow formula. Cell technology and Transwell experiments explore its role in glioma. Then further search for the target gene.DNM3 (Dynamin 3) of miRNA is a member of the superfamily of the promoter protein (including DNM1, DNM2, DNM3), and participates in many membrane transport functions, such as cytokinesis, phagocytosis, transport vesicle buds and cell flow. Recent studies have shown that The high expression of DNM3 inhibits cell proliferation and induces apoptosis. Based on a full analysis of current literature, we speculate that the miRNA-DNM3 pathway may be a therapeutic target for inhibiting the growth of glioma and chemosensitization. As DNM3 appears in the prediction of Targetscan, we further study the relationship with miR-221. We identified the target gene DNM3 of miR-221 and the transcriptional regulator RELA of miR-221, and confirmed by the double luciferase reporter gene experiment, and analyzed the correlation of their expression. Finally, the effect of miR-221 on the glioma was confirmed by the targeting regulation of DNM3 by the virus transfection experiment, and was regulated by the upstream RELA. Control. Part 1 expression of miR-221 in human glioma tissue and GBM derived exocrine and its relationship with pathological grade of glioma WHO: To study the expression of miR-221 in human glioma tissue and GBM derived exocrine, and to analyze the relationship between the expression of miR-221 and the pathological grade of glioma WHO. Methods: 148 patients with glioma and 11 cases of glioma. The tissue specimens of non tumor patients were collected from the Department of Neurosurgery at the second hospital of Hebei Medical University in 2015, including 23 women, 36 men, aged from 30 to 68. The specimens were quickly frozen in liquid nitrogen after collection, and then stored at -80 C. The diagnosis results were guided by 2 pathologists according to the guidance of.2 to extract the above patients. Blood serum, after room temperature statics, obtained serum or immediately after collection, centrifugation to obtain plasma supernatant, extract exocrine.3 SHG-44, U87MG, HEB and U251, and extract.4 quantitative reverse transcriptional polymerase chain reaction (qRT-PCR) method for the determination of glioma tissue or exocrine miR-221 content.5 application of t test and analysis of T analysis of neuroglia The relationship between the miR-221 content of the tumor and the grade of glioma or different cell lines. Results: 1 miRNA-221 was up-regulated in the human glioma tissue. Compared with the control tissue, the expression of miRNA-221 in the glioma tissues was highly expressed (P0.01), and the expression of miRNA-221 in the high malignant glioma tissues was significantly higher than that of the low-grade malignant (P0.01). In WHO The expression of III and WHO IV glioma was significantly higher than that of WHO II or non tumor tissue (P0.01), and the expression of.MiR-221 was significantly correlated with the pathological grade of glioma (WHO grading). The difference of.2 in the expression of miR-221 in the non cellular and plasma exocrine bodies showed significant difference between the miR-221 expression of glioma and non tumor patients (P0). .01) the detection results in.3 plasma were similar to that of tissue detection. Compared with non tumor patients (n=11), the miR-221 expression of plasma exocrine miR-221 was significantly different (P0.01). The expression of miR-221 expression in plasma exocrine miR-221 in glioma patients increased with the pathological grade of glioma significantly increased.4 miR-221 in four gliomas The expression of miR-221 in the Exocyst of U87MG cells was the highest, and the expression of U251, SHG-44 and HEB cells decreased in turn. Conclusion: 1 miR-221 is highly expressed in human glioma tissue, and high expression in the external secretory of plasma source or glioma cell line, and its expression is along with glioma WHO disease. The expression of miR-221 in high grade (III and IV) glioma was significantly higher than that of low grade (II grade) glioma, and the difference was statistically significant positive correlation between the expression of.2 miR-221 and the pathological grade of glioma WHO, indicating that the exocrine miR-221 may be used in the clinical detection of glioma. Second part U87MG cells The effect of source miR-221 on biological activity of SHG-44 cells: To study the effects of U87MG cell derived exocrine miR-221 on proliferation, invasion and chemotherapy resistance of SHG-44 cells. Methods: 1 culture U87MG and SHG-44 cell lines, and the extraction of U87MG cell line exocrine.2 to SHG-44 cell lines for anti-miR-221 (miR-221) Expression of miR-221 in low cell and transfection of anti-miR-NC (Ctrl ASO) as control. In the experimental group SHG-44 cell culture medium also added exosecreting.3 to carry out CCK-8 experimental study to reduce the proliferation ability of glioma cells after miR-221 expression and.4 performed by flow cytometry to reduce the apoptosis ability of miR-221 expression to glioma cells and temozolomide Drug resistance changes.5 conducted Transwell experiment and scratch test to reduce the invasion ability of glioma cells after miR-221 expression. Results: 1 qRT-PCR results showed that after transfection of anti-miR-221, the expression of miR-221 in SHG-44 cells decreased significantly (P0.01).2 CCK-8 experimental results, anti-miR-NC+Exo group was the highest proliferation activity. Anti-miR-2 The proliferation activity of the 21 groups was the lowest, that is, after the expression of low miR-221, the proliferation ability of SHG-44 in glioma cells decreased significantly (P0.01).3 flow cytometry results showed that the apoptosis ability of SHG-44 cells was not significantly changed after the knockout of low miR-221 expression, but the sensitivity to temozolomide increased significantly (P0.01).4 Transwell experiment and scratch test results. The invasion ability of SHG-44 cells decreased significantly (P0.01) after the knockout of miR-221. Conclusion: the proliferation and invasion ability of SHG-44 cells decreased significantly and the apoptosis ability was not changed, but the sensitivity to temozolomide increased, but.MiR-221 could affect the proliferation of glioma cells, invasion and chemotherapy of temozolomide. Resistance. The regulation of the downstream target gene DNM3 by part third miR-221 and the effect of RELA on the transcriptional ability of miR-221: to find the downstream target of miR-221 and the transcriptional regulator of miR-221, and to understand the mechanism of the effect of miR-221 on the biological effects of glioma. Method: 1 to predict the downstream effect of miR-221 with Targetscan database. Target, and collect relevant literature screening meaningful results.2 construction of miR-221 overexpression lentivirus plasmid, miR-221 overexpression of stable SHG-44 cell line, stable cell line screening,.3 double luciferase reporter gene detection is used to verify the relationship between miR-221 and DNM3,.4 application qRT-PCR and Western blot (Western blot) method for detecting M The expression of iR-221 and DNM3 in SHG-44 cells and the correlation of expression.5 through CCK-8 experiments, flow cytometry, Transwell migration and invasion experiments, the effect of up regulation of DNM3 on the proliferation, invasion and apoptosis of glioma SHG-44 cells,.6 through bioinformatics methods, and prediction of miR-221 promoter region The transcriptional factor binding site; using luciferase reporter assay to verify the binding of the transcription factor to the miR-221 promoter in SHG-44 cells. By up or down this transcription factor, the real-time quantitative PCR method was used to detect the changes in the expression of miR-221. Results: 1 according to the prediction results of Targetscan and the related literature, It was found that there were two target sites on DNM3 with miR-221,.2 successfully constructed the miR-221 overexpressed lentivirus and glioma cell lines, and the expression level of miR-221 increased significantly (P0.01), and the results of the 45.5 times.3 double Luciferase Report of the control group showed that miR-221 could significantly inhibit the activity of luciferase (P0.0) compared with miR-NC (P0.0). 1), and the expression of DNM3 in glioma tissues was detected after the mutation target site (P0.01).4 qRT-PCR: the expression of DNM3 expression decreased with the histopathological level of glioma and decreased.Spearman rank correlation analysis, indicating that the expression of DNM3 in glioma cells was negatively correlated with the expression of miR-221 (Spearman r=-0.908, P0.01).5. After the carrier, the expression of DNM3 was low in the cells with high expression of miR-221. The CCK-8 experiment suggested that the proliferation of the cells decreased significantly after the increase of the expression of DNM3. Flow cytometry detected that DNM3 promoted the early apoptosis of glioma cells (P0.01).Transwell experiment and scratch test. The results showed that the transfection of DNM3 was inhibited. The migration and invasion ability of glioma cells.6 predicted the presence of RELA binding sites in the promoter region of the miR-221 encoding gene by bioinformatics method, and the double luciferase reporter gene detection confirmed that RELA could be combined with the promoter region of the miR-221 gene. The real-time quantitative PCR results showed that after transfection of RELA, miR-221 could be induced by the transfection of RELA. Expression. Conclusion: the targeting site of miR-221 in glioma is found in DNM3 mRNA. Transcription factor RELA regulates the transcription of miR-221 gene. Fourth part miR-221 promotes the growth of SHG-44 human glioma cell line in vivo. The objective of the study is to confirm the effect of miR-221 on the biological activity of glioma in vivo. Method: 1 stable transfection of miR-221 SHG- 44 glioma cell lines were screened for stable cell lines. The control group was transfected with miR-NC.2 using reverse transcriptional polymerase chain reaction (qRT-PCR) to detect the expression of miR-221 in SHG-44 cells. The growth of.4 tumor was observed in the subcutaneous human glioma model of.3 nude mice. The length of a tumor was measured every 4 days with a vernier caliper. A) and wide diameter (b). Tumor volume was calculated. Results: 1 the miR-221 overexpressing SHG-44 glioma cell line was successfully constructed, and the stable cells were detected by qRT-PCR.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R739.41

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