肾母细胞瘤异常表达基因的筛选及功能鉴定

发布时间：2018-03-13 19:08

本文选题：肾母细胞瘤　切入点：系统评价　出处：《重庆医科大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景:肾母细胞瘤(Wilms'tumor,WT)是儿童泌尿系统中最常见的恶性肿瘤,发病高峰年龄为1-3岁,约有5%的患儿双侧肾脏受累,年发病率为1/10000。大多数患儿为散发病例,大约有10%WT病例伴发生殖细胞突变和(或)先天性发育畸形,比如Beckwith-Wiedemann、Denys-Drash、Perlman和WAGR等综合征。WT是起源于原始后肾胚基的恶性混合性肿瘤,主要包含上皮、间质和胚芽等成分,除了以上三种典型成分外,WT中也可能包含软骨、骨样组织及神经组织等成分,这些异质性成分提示WT病因的复杂性并激发科研人员数十年的积极探索。WT1作为WT第一个抑癌基因,于1990首次被克隆。随后,CTNNB1、WTX等基因突变相继被发现并克隆。WT1、CTNNB1和WTX基因突变约占到总病例数的1/3。同时,WT的发生发展还与其他许多基因改变有关,比如p53、MYCN、CITED1、SIX2、TOP2A及CRABP2等。尤其是在间变型WT中发现有75%存在p53基因突变,提示该基因突变是组织不良型WT的不良预后标志物,可能与WT进展、复发及转移有密切关系。近年来,相继有多个研究团队报道发现WT中与小RNA相关基因突变(如DROSHA和DGCR8),约15%WT的样本中被检测出。与MYCN基因突变一样,DROSHA和DGCR8等基因突变也只是小众突变基因,还有大量wt的驱动基因的突变尚未明确。目前已有大量拷贝数改变及杂合性丢失(loh)等相关病例报道,其中一些改变如11ploh和17p拷贝数减少已明确影响相关基因,但其他位点如1q拷贝数增加、1p拷贝数减少及16q拷贝数减少等尚不能确认与具体基因相关。maw等于1992年首次报道了16qloh变异频率,并证实部分伴发1ploh。随后nwts-3和nwts-4的两项研究结果表明,1p和(或)16qloh与不良预后存在正相关性,并建议该类患儿需要强化化疗。因此,探讨wt的病因,寻找wt的分子生物学标志物,对儿童wt的诊断、治疗及预后具有重大的价值。过去数十年,已有大量wt相关基因突变频率被报道,但结果常常差异较大,究其原因可能与研究样本量、种族差异及测序方法各异等因素有关。为了克服单个研究的局限性,本课题首先采用系统评价方式,全面阐述与wt相关基因的功能和生物学意义。随后采用新一代测序技术,对wt与癌旁正常组织全转录组高通量测序,筛选相关差异表达基因并进行功能验证。本文的主要研究内容及结果如下:第一部分:wt相关基因变异频率的系统评价目的:基于系统评价,全面分析wt相关基因变异频率及意义。方法:计算机检索pubmeb、ovid、embase和cochranelibrary等数据库,检索时限截至2015年9月。由2名研究员按照纳入与排除标准独立筛查文献、提取信息和评价质量后,应用stata12.0软件进行系统评价。结果:1.wt1、wtx和ctnnb1基因总变异约占总病例数的1/3。2.p53的突变与组织不良型wt有关。3.mycn在wt的发生率并不高,它可能不是wt的驱动基因缺陷。4.drosha、dgcr8等基因改变可能与wt病因有关,但仍需更多大样本实验证实。5.loh在wt病因诊断中敏感性较低,需要结合其他生物学标记分析。第二部分:wt全转录组测序分析目的:构建wtcdna文库,筛选差异表达基因。方法:1.采用高通量深度测序分析法,对wt组织及癌旁正常组织进行全转录组测序。2.q-pcr验证差异表达基因。结果:1.成功构建cdna文库。2.筛选出差异表达候选基因有asic1、scg5、hnrnpl和cacnb4等。3.q-pcr验证asic1、scg5、hnrnpl及cacnb4等差异表达基因与测序结果基本一致。第三部分:hnrnpl在wt增殖、凋亡中的调控作用及机制研究目的:探讨hnrnpl在wt增殖、凋亡中的调控作用及机制研究。方法:1.利用rna免疫共沉淀方法探讨hnrnpl蛋白与p53mrna之间的关系。2.构建sirnahnrnpl表达载体,体外转染g401细胞株,mtt法分析g401细胞生长变化,流式细胞术检测细胞调亡情况。体内裸鼠实验,观察肿瘤生长情况。3.q-pcr和westernblot检测hnrnpl基因被抑制后g401细胞hnrnpl、p53和bcl2mrna和蛋白质的表达。结果:1.rna免疫共沉淀实验证实hnrnpl能与p53的mrna特异性的结合。2.沉默hnrnpl基因后,g401细胞体内外生长抑制、细胞凋亡加速。3.沉默hnrnpl基因后,hnrnpl、p53和bcl2mrna和蛋白质的表达下调。结论:与wt相关的基因改变包括:wt1、wtx、ctnnb1、p53、mycn、drosha、dgcr8等基因,以及多个位点的拷贝数改变及杂合性丢失等。但以上改变频率并不高,目前还有大量驱动基因尚未确认,需要以后进行基因测序研究探明。通过转录组测序成功构建cDNA文库,筛选出差异表达候选基因有ASIC1、SCG5、hnRNPL和CACNB4等。发现hnRNPL在WT中表达上调,并能与p53的mRNA特异性的结合。沉默hnRNPL基因后,hnRNPL、p53和Bcl2的mRNA及蛋白表达下调,G401细胞体内外生长抑制、细胞凋亡加速。hnRNPL可能通过p53/Bcl2信号通路参与WT的增殖与凋亡。
[Abstract]:Background: nephroblastoma (Wilms'tumor, WT) is the most common malignant tumor of urinary system in children, the peak age of onset was 1-3 years old, about 5% of children with bilateral renal involvement, the annual incidence rate of 1/10000. in most cases are sporadic cases, about 10%WT of the patients with germ cell mutation and (or) congenital of malformations, such as Beckwith-Wiedemann, Denys-Drash, Perlman and WAGR.WT syndrome is a malignant mixed tumor originated from primitive metanephric blastema, including epithelial, stromal and germ and other ingredients, in addition to the above three kinds of typical components, including cartilage may WT, osteoid tissue and nerve tissue components the complexity of these heterogeneous elements, suggesting that the etiology of WT and stimulate researchers for decades and actively explore.WT1 as the first WT tumor suppressor gene, was cloned for the first time in 1990. Subsequently, CTNNB1, WTX gene mutations have been found and Cloning of.WT1, CTNNB1 and WTX mutations account for about 1/3. of the total number of cases at the same time, the development of WT and many other related genes, such as p53, MYCN, CITED1, SIX2, TOP2A and CRABP2. Especially in anaplastic WT found 75% mutations of p53, suggesting that the gene mutation is poor prognosis of adverse tissue type WT markers, and WT may progress, there is a close relationship between recurrence and metastasis. In recent years, there have been many research teams reported associated with small RNA gene mutations in WT (such as DROSHA and DGCR8), about 15%WT in the samples were detected. MYCN gene mutation, DROSHA and DGCR8 gene mutation are niche mutation and gene mutation driven a large number of WT is not yet clear. There are a large number of copy number changes and loss of heterozygosity (LOH) and other related reports, including some changes such as 11ploh and 17p copy number has clearly reduced Effect of related genes, but other sites such as 1q copy number increase, the copy number of 1p and 16q reduce the reduction in the number of copies is not confirmed with specific genes related to.Maw in 1992 was reported for the first time 16qloh mutation frequency, and confirmed that part with 1ploh. followed by nwts-3 and nwts-4 of the two study results indicate that 1p and (or) 16qloh there is a positive correlation with poor prognosis, and suggested that these patients need intensive chemotherapy. Therefore, to explore the causes of WT, finding the molecular markers of WT, diagnosis of WT in children, with great value of treatment and prognosis. Over the past few decades, there has been a large number of WT related gene mutation frequency was reported, but the results are often quite different may, the reason and research sample, about racial differences and different factors such as sequencing method. In order to overcome the limitations of individual studies, this paper adopts the system evaluation methods, a comprehensive exposition of wt base The function and biological significance. Then by using a new generation of sequencing technology, WT tissues and normal whole transcriptome high-throughput sequencing, screening of differentially expressed genes and functional verification. The main research contents and results of this paper are as follows: the first part: a systematic review of WT related gene mutation frequency Objective: Based on the evaluation system comprehensive analysis, frequency and significance of WT gene mutation. Methods: We searched pubmeb, Ovid, EMBASE and cochranelibrary databases were searched by the end of September 2015. By 2, according to the inclusion and exclusion criteria independent screening literature, information extraction and quality evaluation, evaluate the application of stata12.0 software. Results: 1.wt1 and WTX. CTNNB1 gene mutation and total variation accounted for about.3.mycn in adverse tissue type wt wt incidence rate is not high the total number of cases of 1/3.2.p53, it may not drive gene wt The defect of.4.drosha, dgcr8 gene may be related to the etiology of WT, but it still needs more large sample tests showed that.5.loh sensitivity in WT diagnosis is low, need to be combined with other biological markers. The second part: WT whole transcriptome sequencing analysis objective: to construct a wtcdna Library of differentially expressed genes. Methods: 1. using high throughput deep sequencing analysis, gene expression of whole transcriptome sequencing.2.q-pcr verification on WT of different tissues and adjacent normal tissues. Results: 1. successfully constructed cDNA.2. library screened differentially expressed candidate genes ASIC1, scg5, hnrnpl and cacnb4.3.q-pcr scg5, hnrnpl ASIC1 verification and cacnb4 of differentially expressed genes and sequencing results consistent. The third part: hnrnpl in WT proliferation, apoptosis objective to study the regulatory role and mechanism of hnrnpl in WT: To investigate the effect and mechanism of proliferation, regulation of apoptosis. Methods: 1. using RN A co immunoprecipitation method to study the relationship between the.2. protein and hnrnpl p53mrna to construct sirnahnrnpl expression vector, transfected g401 cell line, analysis of the growth changes of g401 cell MTT assay, flow cytometry to detect the apoptosis of g401 cells in nude mice. In vivo, hnrnpl detection of hnrnpl gene in tumor growth was observed with.3.q-pcr and Westernblot was inhibited. The expression of p53 and bcl2mrna and protein. Results: 1.rna co immunoprecipitation experiments confirmed that hnrnpl can bind to.2. hnrnpl gene silencing and p53 specific mRNA, g401 cells growth inhibition, apoptosis accelerated.3. hnrnpl gene silencing, hnrnpl, down regulate the expression of p53 and bcl2mrna and protein. Conclusion: WT the genetic changes include: WT1, WTX, CTNNB1, p53, MYCN, Drosha, dgcr8 genes, and the copy number changes of multiple sites and loss of heterozygosity. But the above change frequency is not high At present, there are a large number of driver genes has not been confirmed to be the study of gene sequencing proved by transcriptome sequencing. CDNA library was constructed and screened differentially expressed candidate genes ASIC1, SCG5, hnRNPL and CACNB4. It is found that the upregulation of the expression of hnRNPL in WT, and can be combined with p53 mRNA specific hnRNPL gene silencing. After hnRNPL, mRNA and protein expression of p53 and downregulation of Bcl2, G401 cells growth inhibition, cell apoptosis by.HnRNPL might accelerate the proliferation and apoptosis of p53/Bcl2 signaling pathway in WT.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.11

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