本文關(guān)鍵詞： 膀胱癌 微小RNA 細(xì)胞周期 增殖 FLOT1　出處：《浙江大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：背景膀胱癌是目前泌尿系統(tǒng)最常見的惡性腫瘤之一。雖然局限性膀胱腫瘤能通過外科手術(shù)切除而得以控制,但目前對于膀胱癌術(shù)后的高復(fù)發(fā)和高轉(zhuǎn)移率臨床仍然缺少有效的控制手段。我們對膀胱癌的發(fā)病原因和進展機制所知甚少,除了常見的流行病學(xué)和遺傳學(xué)因素,許多錯綜復(fù)雜的分子事件是導(dǎo)致腫瘤細(xì)胞形成、進展和轉(zhuǎn)移的深層次原因。miRNA是真核生物體內(nèi)一種高度保守的,長度為20-24個核苷酸的非編碼RNA,其在人體組織器官內(nèi)的表達分布具有明顯的時空特異性,不同的組織細(xì)胞、同一生物體的不同發(fā)育階段呈現(xiàn)出不同的表達譜。miRNA參與了細(xì)胞生長、發(fā)育、免疫應(yīng)答、周期、凋亡、衰老、運動等多個生理過程。國內(nèi)外研究者已在包括膀胱癌在內(nèi)的多種腫瘤內(nèi)發(fā)現(xiàn)了異常的miRNA表達譜,腫瘤細(xì)胞內(nèi)相較正常細(xì)胞呈現(xiàn)差異化表達的miRNA,往往在腫瘤的發(fā)生發(fā)展中起到了關(guān)鍵作用,其中低表達的miRNA在腫瘤細(xì)胞中多起到抑癌基因的作用。本研究希望在膀胱癌中尋找異常表達miRNA,并進一步揭示其在腫瘤基因調(diào)控中的作用。目的在收集到的膀胱癌患者手術(shù)切除標(biāo)本中采取實時熒光定量PCR的方法檢測miR-608的表達水平,分析影響miRNA表達的表觀遺傳學(xué)機制。以獲得性功能實驗觀察miR-608對膀胱癌細(xì)胞系T24和UM-UC-3的一系列生物學(xué)功能的影響。以生物信息學(xué)分析為基礎(chǔ),預(yù)測miR-608潛在的的調(diào)控靶點,通過對靶點基因的沉默和過表達實驗,驗證miR-608對靶點調(diào)控的意義,并進一步探索調(diào)控中存在的分子機制。方法1.在13對膀胱癌組織標(biāo)本中檢測miR-608的相對表達量,對膀胱癌組織及與其配對的正常膀胱粘膜上皮組織中的miR-608表達差異進行統(tǒng)計,并對不同病理分期與分級的膀胱癌中miR-608表達水平的差異進行分析。2.通過對T24和UM-UC-3膀胱癌細(xì)胞株體外轉(zhuǎn)染miR-608 mimic(模擬體)的方式來進行一系列獲得性功能實驗。以CCK-8、平板克隆形成、流式細(xì)胞周期和凋亡檢測、劃痕實驗以及Transwell小室遷移和侵襲、活體成瘤實驗來分析miR-608過表達對腫瘤細(xì)胞增殖與侵襲遷移功能的影響。3.利用在線生物信息學(xué)工具,預(yù)測并挑選miR-608作用的靶基因,然后用Western Blot、Real Time PCR等檢測手段在上述細(xì)胞株內(nèi)驗證miR-608預(yù)測結(jié)合的靶點蛋白基因的mRNA及蛋白水平的相應(yīng)變化,通過構(gòu)建螢火蟲熒光素酶雙報告質(zhì)粒對miR-608所作用的靶基因3'-UTR結(jié)合位點進行驗證,最后通過小RNA干擾和過表達拯救等實驗來最終證實miRNA和靶點基因的存在的靶向調(diào)控關(guān)系。結(jié)果1.收集的13例膀胱癌病人的腫瘤組織標(biāo)本中,miR-608的表達水平均顯著降低,相對于配對癌旁組織,膀胱癌組織中miR-608的平均表達量下降了2/3,肌層與非肌層浸潤性膀胱癌以及高級別與低級別膀胱癌之間的miR-608的表達水平顯示出一定的差異,但無顯著的統(tǒng)計學(xué)意義。2.膀胱癌細(xì)胞系T24和UM-UC-3中miR-608的啟動子區(qū)CpG島呈現(xiàn)高度甲基化狀態(tài),提示DNA甲基化可能與miR-608在膀胱癌中的低表達有關(guān)。3.體外轉(zhuǎn)染miR-608 mimic至T24和UM-UC-3細(xì)胞株以過表達miR-608,可明顯抑制其生長。50nM濃度的miR-608 mimic即可明顯抑制膀胱癌細(xì)胞的體外增殖能力,流式細(xì)胞周期檢測發(fā)現(xiàn)miR-608過表達可誘導(dǎo)上述兩種膀胱癌細(xì)胞發(fā)生G1期阻滯,miR-608過表達還可抑制膀胱癌細(xì)胞體外克隆形成能力。同時,miR-608過表達還可顯著抑制UM-UC-3細(xì)胞株在裸鼠體內(nèi)的成瘤能力。細(xì)胞凋亡檢測提示miR-608過表達并不能誘導(dǎo)細(xì)胞凋亡,而Transwell小室和劃痕實驗則提示miR-608抑制膀胱癌細(xì)胞侵襲和遷移能力的作用同樣并不顯著。4.通過在線生物信息學(xué)軟件分析與文獻報道的相關(guān)芯片數(shù)據(jù)結(jié)果相結(jié)合,預(yù)測FLOT1可能是miR-608的作用靶基因。同時,在過表達miR-608后,檢測到的靶基因FLOT1在mRNA水平和蛋白水平都有顯著下調(diào)。進一步的熒光素酶雙報告實驗結(jié)果證實FLOT1是miR-608的直接作用靶點,其3'-UTR中存在miR-608調(diào)控的作用序列。5.通過細(xì)胞信號通路分析,我們發(fā)現(xiàn)在過表達miR-608或FLOT1的小干擾RNA (siFLOTl)之后,檢測到AKT/FOXO3a信號通路和細(xì)胞增殖相關(guān)的關(guān)鍵蛋白的mRNA水平和蛋白水平都發(fā)生了相應(yīng)下調(diào),證明了miR-608靶向FLOT1抑制膀胱癌增殖能力的作用機制,是通過AKT/FOXO3a信號通路得以實現(xiàn)的。結(jié)論1.與配對癌旁組織相比,膀胱癌組織中miR-608的相對表達水平較低,但是本研究未能提示miR-608表達量與膀胱癌的惡性程度相關(guān)；miR-608在膀胱癌當(dāng)中的低表達水平,可能受其啟動子區(qū)CpG島甲基化狀態(tài)的表觀遺傳調(diào)控。2.體外轉(zhuǎn)染mimic過表達miR-608可使膀胱癌細(xì)胞株發(fā)生細(xì)胞周期阻滯進而抑制其增殖能力。同時,miR-608的過表達還削弱了膀胱癌癌細(xì)胞的克隆形成能力,但是對膀胱癌細(xì)胞凋亡及侵襲遷移能力并無明顯影響。3. FLOT1是miR-608的直接作用靶點,miR-608靶向FLOT1影響其下游的AKT/FOX03a信號通路,進而發(fā)揮抑制膀胱癌的增殖能力的作用。
[Abstract]:Background: bladder cancer is one of the most common malignant tumor of urinary system at present. Although the limitations of surgical resection of the bladder tumor can be controlled, but the postoperative bladder cancer with high recurrence rate and metastasis rate of clinical is still lack of effective control methods. The incidence of bladder cancer and progress mechanism is poorly understood, in addition to epidemiological and genetic factors in common, many perplexing the molecular events that lead to tumor cell formation, progression and metastasis of.MiRNA deep reason is the eukaryotic organisms within a highly conserved, non RNA encoding length of 20-24 nucleotides, its expression in human tissues and organs in the distribution has obvious specificity of time and space. Different cells at different developmental stages of the same organism showed different expression profiles of.MiRNA involved in cell growth, development, immune response, apoptosis, cycle time The old, sports and other physiological processes. Researchers at home and abroad has been in a variety of tumors, including bladder cancer found in abnormal miRNA expression profiles in tumor cells compared with normal cells show differences in expression of miRNA, often in tumor development plays a key role in the low expression of miRNA in tumor many cells play a role of tumor suppressor genes. This study hopes to find the abnormal expression of miRNA in bladder cancer, and further to reveal its role in the regulation of tumor gene expression level. Take the method of real-time fluorescence quantitative detection of PCR miR-608 in bladder cancer patients collected specimens, to analyze the effect of miRNA the expression of epigenetic mechanisms. The effect of gain of function experiments to observe the effects of miR-608 on bladder cancer cell lines T24 and UM-UC-3 in a series of biological functions. By bioinformatics analysis based on miR- prediction 608 potential targets, the target gene silencing and overexpression experiments, miR-608 verification regulation of the target point, and further explore the molecular mechanism of regulation in the presence of 1. in 13. The relative expression method of bladder cancer tissue samples of miR-608, differential expression statistics of bladder cancer and paired normal bladder epithelial tissues of miR-608, and the different pathological staging and grading of bladder cancer miR-608 expression of.2. were analyzed by T24 and UM-UC-3 in bladder cancer cell lines in vitro transfection of miR-608 mimic (simulation) way to conduct a series of experiments. In order to gain of function CCK-8 tablet cloning, cell cycle and apoptosis detection, scratch test and Transwell chamber migration and invasion in vivo tumorigenicity assay to analyze the expression of miR-608 on invasion and migration of tumor cell proliferation. The influence of.3. using bioinformatics tools, target gene prediction and selection of miR-608 function, and then use the Western Blot Real, Time PCR and other means of detection verified corresponding changes prediction of miR-608 mRNA and protein level of target gene with the above cells, the target gene 3'-UTR construct firefly luciferase double report the effect of plasmid miR-608 binding sites to verify, finally by RNA interference and overexpression of rescue experiments to finally confirmed the presence of target miRNA and target gene regulation to the relationship. The results of 13 cases of bladder cancer patients collected 1. tumor tissue samples, the expression level of miR-608 decreased significantly, compared with paired paracancerous tissue, the average expression of miR-608 in bladder cancer decreased by 2/3, between the muscle and non muscle invasive bladder cancer and high grade and low grade bladder cancer miR-608 The expression level showed some differences, but no significant statistical significance of miR-608.2. in bladder cancer cell lines T24 and UM-UC-3 in CpG island in the promoter region showed a high degree of methylation, suggesting that DNA methylation and miR-608 in bladder cancer in the lower expression of.3. in vitro transfection of miR-608 mimic into T24 and UM-UC-3 cell lines to the expression of miR-608 in vitro can obviously inhibit the growth of.50nM concentration of miR-608 mimic can inhibit bladder cancer cells, detect the overexpression of miR-608 could induce G1 arrest of the two bladder cancer cells by flow cytometry cell cycle, overexpression of miR-608 can inhibit bladder cancer cells in vitro colony formation ability. At the same time, miR-608 the expression also significantly inhibited UM-UC-3 cell tumorigenic ability in nude mice. Cell apoptosis detection indicated that the overexpression of miR-608 did not induce apoptosis and Transwell cell The related chip data and scratch test suggested that miR-608 inhibited the invasion and migration of bladder cancer cells also were not significant.4. by bioinformatics software analysis and literature combining the results of prediction of FLOT1 may be the target genes of miR-608. At the same time, the expression of miR-608, FLOT1 target genes are detected significantly decreased at mRNA and protein level. Dual luciferase assays further confirmed that FLOT1 is a direct target of miR-608,.5. miR-608 sequences in 3'-UTR cells by regulating the signal path analysis, we found that over expression of small interfering RNA miR-608 or FLOT1 (siFLOTl), detected key protein AKT/FOXO3a signaling pathway and cell proliferation in the mRNA and protein level was reduced, proved that miR-608 targeting FLOT1 inhibits bladder cancer. Mechanism of colonization ability, is realized through AKT/FOXO3a signal pathway. Conclusion: 1. compared with paired noncancerous tissues, miR-608 in bladder cancer tissue relative expression level is low, but this study does not suggest that miR-608 expression and malignant degree of bladder cancer; miR-608 in bladder cancer among the low expression level may be affected by the the start of methylation of the CpG promoter region of the epigenetic regulation of.2. transfected mimic over expression of miR-608 resulted in cell cycle arrest mutants inhibit the proliferation of bladder cancer cells. Meanwhile, overexpression of miR-608 also weakened the clone forming ability of bladder cancer cells, but on bladder cancer cell apoptosis and invasion there is no obvious effect of.3. ability FLOT1 is a direct target of miR-608, miR-608 targeting FLOT1 AKT/FOX03a signaling pathway and its downstream, and then inhibit the bladder cancer proliferation The effect of force.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R737.14

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