miR-101抑制膀胱癌进展的作用及其分子机制研究

发布时间：2018-02-20 16:11

本文关键词： 膀胱癌微小RNA 迁移与侵袭 c-Met　出处：《浙江大学》2015年博士论文　论文类型：学位论文

【摘要】：背景：膀胱癌是泌尿系统最常见的恶性肿瘤,也是全身十大常见肿瘤之一。占我国泌尿生殖系肿瘤发病率的第一位,在西方其发病率仅次于前列腺癌,居第2位。然而,目前的研究仍然对膀胱癌的发生以及进展的机制缺乏足够深入的理解。现在可以明确的是,膀胱癌发生及进展是一个多因素涉及、多基因参与、多步骤形成以及多途径变化的过程,异常基因型的基础在外在高危因素的作用下逐渐积累,最终出现表型的恶形。miRNA是近年来研究比较热点的一类由内源基因编码的非编码RNA单链分子,长度约为20-24个核苷酸。人们已经认识到这些普遍存在的小分子在真核基因表达调控中有着广泛的作用。miRNA的水平在不同组织、不同发育阶段中有显著差异,呈现具有分化的位相性和时序性。对于各类疾病,尤其是肿瘤而言,miRNA也出现了时间和空间上的表达异常,本研究希望在膀胱癌中寻找这样的异常表达,并发现其在基因调控中扮演的作用。目的：在膀胱癌患者手术切除的标本组织中采用Real Time PCR的方法检测miR-101的表达情况；体外实验中根据一系列实验方法鉴定miR-101在膀胱癌T24细胞株中的生物学功能。并且,通过生物信息学对miR-101作用靶点进行预测,并行生物学实验验证。方法：1.在20对配对的膀胱癌组织标本检测miR-101的相对表达量,对膀胱癌组织及对照的正常粘膜组织中miR-101的表达差异进行统计,并分析不同病例级别与分期的膀胱癌中miR-101的表达情况； 2.使用对膀胱癌T24细胞株转染miR-101mimic使其过表达的方法进行一系列功能实验。通过CCK-8、平板克隆形成、流式细胞周期和凋亡检测、划痕愈合实验和Transwell小室法等手段来分析上调miR-101的表达对细胞增殖及迁移侵袭能力带来的影响； 3.使用在线软件对miR-101进行靶基因预测,然后利用Real Time PCR, Western Blot等验证靶基因mRNA以及蛋白表达量是否相应变化,并利用荧光素酶双报告系统对miR-101的靶序列进行鉴定,使用拯救实验进一步验证miR-101的作用靶基因。结果：1.收集的20例膀胱癌癌组织标本中,有19例为miR-101相对低表达,占95%。癌组织中miR-101表达量平均约癌旁组织的1/2。肌层浸润性膀胱癌与非肌层浸润性膀胱癌以及高级别膀胱癌与低级别膀胱癌在miR-101的相对表达量的平均值有差异,但未能有统计学上的显著性意义。 2.膀胱癌T24细胞株和正常的膀胱移行上皮细胞株SV-HUC-1相比,miR-101也表现为低表达。 3.体外转染miR-101mimic至膀胱癌T24细胞株使miR-101过表达,50nM浓度可以抑制细胞增殖活力。体外平板克隆形成实验未能明显抑制细胞克隆形成。划痕愈合实验和Transwell小室等实验显示miR-101过表达可以减弱T24细胞的迁移和侵袭能力,但对细胞周期和凋亡无明显影响。 4.通过在线软件的分析,预测c-Met可能是miR-101的靶基因,同时通过Real Time PCR和Western Blot实验检测到c-Met在过表达miR-101后mRNA水平和蛋白水平都有下调。接下来的荧光素酶双报告系统分析证实c-Met是miR-101的直接作用靶点,其3'-UTR中存在miR-101调控的作用序列。结论：1.膀胱癌组织中miR-101与癌旁对照组织相比,相对表达量较低。但是本研究未能提示miR-101表达量高低与膀胱癌的恶性程度相关。 2.体外转染miR-101mimic使miR-101过表达削弱了膀胱癌T24细胞株的增殖能力,但对克隆形成及细胞周期凋亡无明显影响。 3.miR-101的直接靶基因是c-Met,并影响下游ERK1/2蛋白的磷酸化水平。
[Abstract]:Background: bladder cancer is the most common malignant tumor of urinary system, the body is also one of the ten most common tumor. Our first urogenital tumor incidence in the west, its incidence after prostate cancer, ranking second. However, the current study is still on the occurrence of bladder cancer and the mechanism of the lack of progress have a deep understanding. It is now clear that the occurrence and progression of bladder cancer is related to multiple factors, multiple genes, multiple steps and multiple ways to change the formation process, abnormal genotypes accumulated in the external risk factors under the action of ultimate evil shape.MiRNA phenotype is comparative research in recent years focus of a class from non RNA single molecule encoding endogenous gene encoding, the length is about 20-24 nucleotides. It has been recognized that these ubiquitous small molecule regulation is widely expressed in eukaryotic gene The effect of the level of.MiRNA in different tissues, there is significant difference in different developmental stages, showing the spatial and temporal differentiation. For various diseases, especially tumor, the expression of miRNA also appeared on time and space anomalies, this study hopes to find the abnormal expression in bladder cancer, and found it plays a role in gene regulation.
Objective: to detect the expression of miR-101 by Real PCR Time in patients with bladder cancer tissues; in vitro according to a series of experimental methods for identification of miR-101 in bladder cancer cell line T24 in biological function. Moreover, by means of bioinformatics to predict the targets of miR-101, parallel biological experiments.
Methods: 1.. In 20 pairs of paired bladder cancer tissues, the relative expression of miR-101 was detected. The difference of miR-101 expression between bladder cancer tissues and normal mucosa tissues was statistically analyzed, and the expression of miR-101 in different grade and stage of bladder cancer was analyzed.
Methods 2. of bladder carcinoma cell line T24 transfected with miR-101mimic and its over expression of a series of functional experiment. Through CCK-8, colony formation, flow cytometry cell cycle and apoptosis detection, expression means wound healing assay and Transwell chamber method to analyze the regulation of miR-101 brings on the invasion ability of cell proliferation and migration;
3. the use of online software to predict target gene of miR-101, and then use the Real Time PCR, Western Blot mRNA and protein expression of target genes to verify whether the amount of the corresponding changes, and the target sequence of miR-101 was identified by dual luciferase system, using the save target experiment to further validate the miR-101.
Results: 20 cases of carcinoma of bladder cancer specimens collected in 1., there were 19 cases of low miR-101 expression, 95%. miR-101 expression in carcinomas of 1/2. muscle layer averaged about cancer tissues of invasive bladder cancer and non muscle invasive bladder cancer and high-grade bladder cancer and bladder cancer at a relatively low level expression of miR-101 with different average values, but not statistically significant.
2. the T24 cell line of bladder cancer and the normal bladder transitional cell line SV-HUC-1, miR-101 also showed low expression.
3. in vitro transfection of miR-101mimic to bladder cancer cell line T24. Overexpression of miR-101, the concentration of 50nM can inhibit cell proliferation in vitro. Colony formation assay failed to inhibit the formation of cell clones. Wound healing assay and Transwell chamber experiment showed that overexpression of miR-101 can reduce the migration and invasion of T24 cells, but had no obvious effect on cell cycle and apoptosis.
4. through the analysis of online software, c-Met may predict the target gene of miR-101, while Time PCR and Western Real by Blot assay after overexpression of miR-101 to c-Met in mRNA and protein level were down regulated. The dual luciferase system analysis confirmed that c-Met is a direct target of miR-101, miR-101 regulatory sequences the 3'-UTR.
Conclusion: 1., the expression of miR-101 in bladder cancer tissue is lower than that in adjacent tissues. However, this study failed to indicate that the expression level of miR-101 is correlated with the malignancy of bladder cancer.
2. in vitro transfection of miR-101mimic caused miR-101 overexpression to weaken the proliferation ability of bladder cancer T24 cell line, but had no significant effect on clonogenic formation and cell cycle apoptosis.
The direct target gene of 3.miR-101 is c-Met, which affects the phosphorylation level of the downstream ERK1/2 protein.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.14

【共引文献】