發(fā)布時(shí)間：2018-04-09 10:33

  本文選題：microRNA-7　切入點(diǎn)：XIAP　出處：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：第一部分 惡性膠質(zhì)瘤細(xì)胞TRAIL敏感性相關(guān)的microRNA的篩選、功能鑒定及機(jī)制研究【背景】惡性膠質(zhì)母細(xì)胞瘤(GBM)是最具侵襲性的腦腫瘤之一,中位生存時(shí)間不超過(guò)16個(gè)月。盡管對(duì)于它的治療已取得很大的進(jìn)展,但它仍然是最具挑戰(zhàn)性的疾病之一。TRAIL(TNF相關(guān)的凋亡誘導(dǎo)配體)能夠特異性地殺傷腫瘤而不影響正常細(xì)胞,因此是很有潛力的抗癌藥物。但是已有研究表明,許多腫瘤對(duì)其耐受,且在臨床試驗(yàn)中,TRAIL因其半衰期短以及體內(nèi)的不穩(wěn)定致使其實(shí)際應(yīng)用受到了很大限制。然而,最近的研究表明,使用間充質(zhì)干細(xì)胞(MSCs)表達(dá)TRAIL可將TRAIL特異性地運(yùn)送到腫瘤局部,這可在一定程度上補(bǔ)償這一缺點(diǎn)。MicroRNA,一類在進(jìn)化上高度保守的非編碼小RNA,可以通過(guò)與靶基因的3'非翻譯區(qū)(UTRs)結(jié)合發(fā)揮轉(zhuǎn)錄后調(diào)控作用,進(jìn)而抑制mRNA翻譯或降解mRNA。越來(lái)越多的證據(jù)表明,miRNA在腫瘤細(xì)胞的惡性表型的調(diào)控中發(fā)揮著關(guān)鍵作用,而且已有研究證實(shí),細(xì)胞可以外泌體的形式分泌miRNA,進(jìn)而下調(diào)受體細(xì)胞中靶基因的表達(dá)。因此,這提示我們,聯(lián)合使用MSCs、microRNA、TRAIL進(jìn)行腫瘤靶向治療的策略或許會(huì)有很好的治療前景�！灸康摹空页隹梢栽雒鬞RAIL的microRNA,并使用MSCs作為運(yùn)載工具開(kāi)展靶向治療�！痉椒ā繛榱苏业皆谀z質(zhì)瘤中可增敏TRAIL的關(guān)鍵microRNA,我們首先對(duì)對(duì)TRAIL表現(xiàn)不同敏感程度的膠質(zhì)瘤細(xì)胞系進(jìn)行mi RNA表達(dá)譜的分析,從中找到與TRAIL誘導(dǎo)的凋亡呈現(xiàn)顯著相關(guān)性的miRNAs。之后在不同的細(xì)胞中過(guò)表達(dá)或干涉該microRNA,檢測(cè)TRAIL誘導(dǎo)細(xì)胞凋亡的情況,進(jìn)而確認(rèn)增敏TRAIL的關(guān)鍵microRNA。然后通過(guò)生物信息學(xué)預(yù)測(cè)、雙熒光素酶報(bào)告基因?qū)嶒?yàn)、實(shí)時(shí)定量PCR和蛋白印跡實(shí)驗(yàn),找到該miRNA的下游靶基因。同時(shí),通過(guò)功能缺失和恢復(fù)實(shí)驗(yàn),進(jìn)一步確定這樣一條調(diào)控軸線。然后對(duì)MSCs進(jìn)行改造,使其攜帶TRAIL和該microRNA,與膠質(zhì)瘤細(xì)胞系U87進(jìn)行體外共培養(yǎng)實(shí)驗(yàn),驗(yàn)證基因修飾后的MSCs是否可以顯著殺傷腫瘤細(xì)胞。同時(shí),利用外泌體的抑制劑來(lái)進(jìn)行分析,這種影響是否通過(guò)外泌體介導(dǎo)的分子傳遞來(lái)完成的。之后分別通過(guò)transwell實(shí)驗(yàn)和免疫組化實(shí)驗(yàn)在體外和體內(nèi)實(shí)驗(yàn)中分析,攜帶TRAIL和microRNA的MSCs是否具有較好的腫瘤趨向性。最后建立裸鼠皮下荷瘤模型,使用TRAIL和microRNA聯(lián)合過(guò)表達(dá)的MSCs進(jìn)行尾靜脈治療實(shí)驗(yàn),確認(rèn)治療效果�！窘Y(jié)果】我們發(fā)現(xiàn),miR-7在膠質(zhì)瘤細(xì)胞系中的表達(dá)情況與TRAIL誘導(dǎo)的細(xì)胞凋亡存在顯著的正相關(guān)關(guān)系。之后,功能缺失和恢復(fù)實(shí)驗(yàn)證實(shí),miR-7確實(shí)是膠質(zhì)瘤細(xì)胞系中增敏TRAIL關(guān)鍵分子。隨后,在機(jī)制研究中,我們發(fā)現(xiàn),XIAP是其介導(dǎo)該作用的關(guān)鍵的下游靶基因,進(jìn)一步的研究發(fā)現(xiàn),miR-7-XIAP這種調(diào)控關(guān)系存在于多種腫瘤中。而且,體內(nèi)體外實(shí)驗(yàn)證實(shí),攜帶TRAIL和microRNA-7的間充質(zhì)干細(xì)胞具備很好的腫瘤趨向性。同時(shí),通過(guò)外泌體抑制實(shí)驗(yàn)證實(shí),MSCs可以以外泌體的形式分泌microRNA-7進(jìn)而下調(diào)受體細(xì)胞中的XIAP,從而增強(qiáng)受體細(xì)胞的凋亡。最后,更重要的是,在裸鼠移植瘤的治療模型中,TRAIL和microRNA-7聯(lián)合過(guò)表達(dá)的間充質(zhì)干細(xì)胞表現(xiàn)出很好的促凋亡作用,從而抑制腫瘤的生長(zhǎng),達(dá)到較好的治療效果�！窘Y(jié)論】在腫瘤細(xì)胞中,miR-7可以通過(guò)下調(diào)XIAP顯著增強(qiáng)TRAIL誘導(dǎo)的細(xì)胞凋亡。攜帶TRAIL和miR-7的MSCs可以特異性地趨向于腫瘤局部,抑制腫瘤的生長(zhǎng)。第二部分 microRNA-7在肝癌細(xì)胞中的抑癌新機(jī)制研究【背景】Micro RNA(miRNA)分子是一類在多細(xì)胞生物中廣泛轉(zhuǎn)錄的、普遍存在的非編碼小RNA分子。成熟的mi RNA分子可通過(guò)序列特異性的堿基互補(bǔ)配對(duì)結(jié)合到m RNA的3’非編碼區(qū),形成RNA沉默復(fù)合物,進(jìn)而導(dǎo)致靶m RNA的翻譯抑制或直接將其降解。更重要的是,一個(gè)mi RNA可以同時(shí)調(diào)節(jié)上百種m RNA,這也使得其能夠參與錯(cuò)綜復(fù)雜的分子調(diào)控網(wǎng)絡(luò),進(jìn)而影響細(xì)胞的生命活動(dòng)。越來(lái)越多的研究表明,mi RNA的正常表達(dá)是生命活動(dòng)正常運(yùn)轉(zhuǎn)所必需的,而它的失調(diào)可能會(huì)導(dǎo)致包括腫瘤在內(nèi)的多種疾病的發(fā)生。近來(lái)研究發(fā)現(xiàn),在肝癌的發(fā)生發(fā)展過(guò)程中伴隨著一些抑癌mi RNA的下調(diào),而在這些mi RNA中,mi R-7在肝癌組織中顯著低表達(dá),且mi R-7的過(guò)表達(dá)能夠抑制肝癌的增殖和轉(zhuǎn)移。也有研究證實(shí),mi R-7可以通過(guò)靶向PI3K/AKT通路抑制肝癌的生長(zhǎng)和轉(zhuǎn)移。但是,這些都還不能充分揭示mi R-7在肝癌中發(fā)揮的關(guān)鍵抑癌作用�！灸康摹筷U明microRNA-7在HCC細(xì)胞中的新的抑癌機(jī)制�！痉椒ā渴紫�,在肝癌細(xì)胞中瞬時(shí)轉(zhuǎn)染miR-7,48小時(shí)后使用流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期。之后,通過(guò)生物信息學(xué)預(yù)測(cè)工具分析預(yù)測(cè)mi R-7的可能下游靶基因,隨后通過(guò)雙熒光素酶報(bào)告基因?qū)嶒?yàn)初步確認(rèn)mi R-7的下游靶基因。然后通過(guò)實(shí)時(shí)定量PCR和Western blot檢測(cè)mi R-7過(guò)表達(dá)后的靶基因的m RNA和蛋白表達(dá)水平的變化,同時(shí)通過(guò)功能缺失和恢復(fù)實(shí)驗(yàn)進(jìn)一步確認(rèn)靶基因與細(xì)胞周期的關(guān)系。最后在腫瘤臨床樣品和腫瘤細(xì)胞系中分析mi R-7與靶基因的RNA表達(dá)水平的相關(guān)性�！窘Y(jié)果】首先,我們發(fā)現(xiàn),在肝癌細(xì)胞中過(guò)表達(dá)miR-7可以顯著導(dǎo)致細(xì)胞周期G1期阻滯。之后通過(guò)生物信息學(xué)預(yù)測(cè)、報(bào)告基因?qū)嶒?yàn)、實(shí)時(shí)定量PCR和蛋白印跡實(shí)驗(yàn)證實(shí),細(xì)胞周期蛋白CCNE1是mi R-7發(fā)揮該抑制作用的關(guān)鍵下游靶基因。進(jìn)一步的研究證實(shí),干涉CCNE1與過(guò)表達(dá)mi R-7導(dǎo)致相似的細(xì)胞周期變化,同時(shí)CCNE1的恢復(fù)過(guò)表達(dá)可以消除mi R-7引起的細(xì)胞周期G1期阻滯。最后,臨床組織樣品和細(xì)胞系的結(jié)果顯示,mi R-7與CCNE1的表達(dá)水平存在著顯著的負(fù)相關(guān)關(guān)系�！窘Y(jié)論】miR-7可以直接靶向下調(diào)細(xì)胞周期蛋白CCNE1的表達(dá),進(jìn)而導(dǎo)致細(xì)胞周期G1期阻滯,從而抑制肝癌細(xì)胞增殖。
[Abstract]:The first part of the screening of malignant glioma microRNA cells related to TRAIL sensitivity, function identification and mechanism study [background] malignant glioblastoma (GBM) is one of the most aggressive brain cancer, the median survival time of less than 16 months. Although the treatment has made great progress, but it is still one of the most challenging disease.TRAIL (TNF related apoptosis inducing ligand) can specifically kill tumor cells without affecting normal, so it is a potential anticancer drug. But studies have shown that the tolerance of many tumors, and in clinical trials, TRAIL because of its short half-life and in vivo the instability resulting in its practical application is limited. However, recent studies suggest that the use of mesenchymal stem cells (MSCs) expression of TRAIL TRAIL can be specifically delivered to the tumor, which can be compensated to a certain extent. The disadvantages of.MicroRNA, a class of evolutionarily conserved non small RNA encoding, with target gene 3'untranslated region (UTRs) combines post transcriptional regulation, thereby inhibiting translation or degradation of mRNA. mRNA growing evidence that miRNA plays a key role in the regulation of tumor cell malignant phenotype in, and studies have confirmed that cells can form the exosome secretion of miRNA, and down regulate the expression of receptor cells in target genes. Therefore, this indicates that the combined use of MSCs, microRNA, TRAIL of tumor targeted therapy strategies may have good prospects for treatment. [Objective] to find out can increase sensitive TRAIL microRNA, and uses MSCs as a vehicle to carry out targeted therapy. [method] in order to find the key of microRNA in glioma can be sensitized by TRAIL, we first on TRAIL performance of different sensitivity of glioma fine Cell lines mi RNA expression profiles, from apoptosis induced by TRAIL and found a significant correlation between the expression of miRNAs. or the microRNA interference in different cells, detect the apoptosis induced by TRAIL, and then confirm the key microRNA. sensitized TRAIL and then through bioinformatics prediction, dual luciferase reporter gene the experiment, real time quantitative PCR and Western blot to find the downstream target gene of miRNA. At the same time, the loss of function and recovery experiments, further confirmed such a regulation for the transformation of the MSCs axis. Then, the carrying TRAIL and the microRNA, and in vitro U87 glioma cell line co culture experiments, verify whether gene the modified MSCs can effectively kill tumor cells. At the same time, with the analysis of the molecular inhibitors of exosomes, whether this effect by exosome mediated transfer to complete . after respectively by Transwell test and immunohistochemical experiments in vitro and in vivo experiments, whether carrying TRAIL and microRNA MSCs have good tumor tropism. Finally, establish the model of tumor bearing nude mice, using TRAIL and microRNA combined with MSCs over expression of caudal vein treatment experiment, confirm the therapeutic effect. [results] we found that there was a significant positive correlation between miR-7 apoptosis in glioma cell lines and the expression induced by TRAIL. After the loss of function and recovery experiments confirmed that miR-7 is indeed sensitized TRAIL key molecules in glioma cell lines. Then, in the mechanism study, we found that XIAP is a downstream target the key genes mediating this effect, further research found that the miR-7-XIAP of this regulation relationship exists in a variety of tumors. Moreover, confirmed in vivo and in vitro experiments, carrying TRAIL and microRNA-7 between Mesenchymal stem cells have good tumor tropism. At the same time, through the exosome inhibition experiments confirmed that MSCs can form outside the urinary secretion of microRNA-7 and down-regulation of receptor XIAP in the cells, thereby enhancing apoptosis of receptor cells. Finally, more importantly, in the treatment model in nude mice, and TRAIL microRNA-7 co expression of mesenchymal stem cells showed apoptosis promoting effect is very good, to inhibit the growth of tumor, achieve better therapeutic effects. [Conclusion] in tumor cells, miR-7 can downregulate XIAP enhanced TRAIL induced apoptosis with TRAIL and miR-7. MSCs can specifically tend to the local tumor, inhibit the growth of tumor. The second part of microRNA-7 in HCC tumor suppressor mechanism study [background] Micro RNA (miRNA) is a class of molecules in multicellular organisms are ubiquitous transcription. Non small RNA molecules. Mi encoding mature RNA molecules by sequence specific base pairing with m RNA 3 'non encoding region, the formation of RNA silencing complex, leading to the target m RNA translational repression or directly degrade. More importantly, a mi RNA can be adjusted at the same time hundreds of M RNA, which was able to participate in the molecular regulation of network and perplexing, cellular activity. More and more studies show that the normal expression of MI RNA is necessary for the normal operation of life activity, and its imbalance may lead to various diseases including tumors. Recent research found that and in the occurrence and development of hepatocellular carcinoma with tumor suppressor mi downregulation of RNA, and in these mi RNA, MI R-7 in hepatocellular carcinoma was significantly lower expression of MI and overexpression of R-7 can inhibit the proliferation and metastasis of hepatocellular carcinoma. There is also evidence Actually, MI R-7 can inhibit the growth and metastasis of liver cancer through the PI3K/AKT pathway to the target. However, these are not fully reveal the key mi R-7 play in hepatocellular carcinoma and inhibition of cancer. [Objective] to elucidate microRNA-7 in HCC cells of the new tumor suppressor mechanism. [method] firstly, transient transfection in hepatocellular carcinoma cells miR-7,48 hours after the cell cycle was detected by flow cytometry. Then through bioinformatics prediction possible downstream target gene prediction analysis tool R-7 MI, then the preliminary confirmation of downstream target gene mi R-7 by dual luciferase assay. Then the expression of M RNA and protein expression of target genes and quantitative real-time PCR Western blot detection of MI after R-7, at the same time through the loss of function and recovery experiments to further confirm the relationship between target gene and cell cycle. Finally, in clinical samples and tumor cell lines in Mi analysis of R-7 and target gene expression level of RNA gene. [results] first, we found that overexpression of miR-7 could obviously induce G1 cell cycle arrest in HepG2 cells. Through bioinformatics prediction, reporter gene assay, confirmed by quantitative real-time PCR and Western blotting experiments, cyclin CCNE1 is a key downstream the target gene mi R-7 play the role of inhibition. Further studies showed that CCNE1 interference and overexpression of MI R-7 leads to cell cycle changes similar to that at the same time the recovery of CCNE1 overexpression can eliminate the G1 phase of the cell cycle blocking Mi induced by R-7. Finally, the clinical tissue samples and cell lines showed that the expression level of Mi R-7 with CCNE1 there is a significant negative correlation. [Conclusion] miR-7 can directly target the expression of cyclin CCNE1, which leads to G1 cell cycle arrest, thereby inhibiting liver cancer cell Cell proliferation.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.41
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本文編號(hào)：1726065