miR-608在膀胱癌中的抑癌作用及其分子机制研究

发布时间：2018-01-26 23:47

本文关键词： 膀胱癌微小RNA 细胞周期增殖 FLOT1　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：背景膀胱癌是目前泌尿系统最常见的恶性肿瘤之一。虽然局限性膀胱肿瘤能通过外科手术切除而得以控制,但目前对于膀胱癌术后的高复发和高转移率临床仍然缺少有效的控制手段。我们对膀胱癌的发病原因和进展机制所知甚少,除了常见的流行病学和遗传学因素,许多错综复杂的分子事件是导致肿瘤细胞形成、进展和转移的深层次原因。miRNA是真核生物体内一种高度保守的,长度为20-24个核苷酸的非编码RNA,其在人体组织器官内的表达分布具有明显的时空特异性,不同的组织细胞、同一生物体的不同发育阶段呈现出不同的表达谱。miRNA参与了细胞生长、发育、免疫应答、周期、凋亡、衰老、运动等多个生理过程。国内外研究者已在包括膀胱癌在内的多种肿瘤内发现了异常的miRNA表达谱,肿瘤细胞内相较正常细胞呈现差异化表达的miRNA,往往在肿瘤的发生发展中起到了关键作用,其中低表达的miRNA在肿瘤细胞中多起到抑癌基因的作用。本研究希望在膀胱癌中寻找异常表达miRNA,并进一步揭示其在肿瘤基因调控中的作用。目的在收集到的膀胱癌患者手术切除标本中采取实时荧光定量PCR的方法检测miR-608的表达水平,分析影响miRNA表达的表观遗传学机制。以获得性功能实验观察miR-608对膀胱癌细胞系T24和UM-UC-3的一系列生物学功能的影响。以生物信息学分析为基础,预测miR-608潜在的的调控靶点,通过对靶点基因的沉默和过表达实验,验证miR-608对靶点调控的意义,并进一步探索调控中存在的分子机制。方法1.在13对膀胱癌组织标本中检测miR-608的相对表达量,对膀胱癌组织及与其配对的正常膀胱粘膜上皮组织中的miR-608表达差异进行统计,并对不同病理分期与分级的膀胱癌中miR-608表达水平的差异进行分析。2.通过对T24和UM-UC-3膀胱癌细胞株体外转染miR-608 mimic(模拟体)的方式来进行一系列获得性功能实验。以CCK-8、平板克隆形成、流式细胞周期和凋亡检测、划痕实验以及Transwell小室迁移和侵袭、活体成瘤实验来分析miR-608过表达对肿瘤细胞增殖与侵袭迁移功能的影响。3.利用在线生物信息学工具,预测并挑选miR-608作用的靶基因,然后用Western Blot、Real Time PCR等检测手段在上述细胞株内验证miR-608预测结合的靶点蛋白基因的mRNA及蛋白水平的相应变化,通过构建萤火虫荧光素酶双报告质粒对miR-608所作用的靶基因3'-UTR结合位点进行验证,最后通过小RNA干扰和过表达拯救等实验来最终证实miRNA和靶点基因的存在的靶向调控关系。结果1.收集的13例膀胱癌病人的肿瘤组织标本中,miR-608的表达水平均显著降低,相对于配对癌旁组织,膀胱癌组织中miR-608的平均表达量下降了2/3,肌层与非肌层浸润性膀胱癌以及高级别与低级别膀胱癌之间的miR-608的表达水平显示出一定的差异,但无显著的统计学意义。2.膀胱癌细胞系T24和UM-UC-3中miR-608的启动子区CpG岛呈现高度甲基化状态,提示DNA甲基化可能与miR-608在膀胱癌中的低表达有关。3.体外转染miR-608 mimic至T24和UM-UC-3细胞株以过表达miR-608,可明显抑制其生长。50nM浓度的miR-608 mimic即可明显抑制膀胱癌细胞的体外增殖能力,流式细胞周期检测发现miR-608过表达可诱导上述两种膀胱癌细胞发生G1期阻滞,miR-608过表达还可抑制膀胱癌细胞体外克隆形成能力。同时,miR-608过表达还可显著抑制UM-UC-3细胞株在裸鼠体内的成瘤能力。细胞凋亡检测提示miR-608过表达并不能诱导细胞凋亡,而Transwell小室和划痕实验则提示miR-608抑制膀胱癌细胞侵袭和迁移能力的作用同样并不显著。4.通过在线生物信息学软件分析与文献报道的相关芯片数据结果相结合,预测FLOT1可能是miR-608的作用靶基因。同时,在过表达miR-608后,检测到的靶基因FLOT1在mRNA水平和蛋白水平都有显著下调。进一步的荧光素酶双报告实验结果证实FLOT1是miR-608的直接作用靶点,其3'-UTR中存在miR-608调控的作用序列。5.通过细胞信号通路分析,我们发现在过表达miR-608或FLOT1的小干扰RNA (siFLOTl)之后,检测到AKT/FOXO3a信号通路和细胞增殖相关的关键蛋白的mRNA水平和蛋白水平都发生了相应下调,证明了miR-608靶向FLOT1抑制膀胱癌增殖能力的作用机制,是通过AKT/FOXO3a信号通路得以实现的。结论1.与配对癌旁组织相比,膀胱癌组织中miR-608的相对表达水平较低,但是本研究未能提示miR-608表达量与膀胱癌的恶性程度相关；miR-608在膀胱癌当中的低表达水平,可能受其启动子区CpG岛甲基化状态的表观遗传调控。2.体外转染mimic过表达miR-608可使膀胱癌细胞株发生细胞周期阻滞进而抑制其增殖能力。同时,miR-608的过表达还削弱了膀胱癌癌细胞的克隆形成能力,但是对膀胱癌细胞凋亡及侵袭迁移能力并无明显影响。3. FLOT1是miR-608的直接作用靶点,miR-608靶向FLOT1影响其下游的AKT/FOX03a信号通路,进而发挥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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.14

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