DAL-1调控非小细胞肺癌EMT的分子机制及其生物信息学研究

发布时间：2018-07-25 19:37

【摘要】：研究背景:目前肺癌是全球发病率和致死率最高的恶性肿瘤,其中非小细胞肺癌(Non-small cell lung cancer,NSCLC)占全部肺癌的80%。晚期NSCLC预后差,侵袭和转移是其主要的致死原因。因此,研究NSCLC转移和侵袭的分子机理、探索有关的分子靶标,成为目前亟待解决的课题。研究目的:本课题重点研究DAL-1基因在NSCLC侵袭与迁移中的作用机理,以希望为NSCLC的临床治疗提供新的靶标与理论基础。研究方法:根据人类DAL-mRNA编码序列,设计并合成靶标DAL-1的3条特异性DAL-1 shRNA表达质粒(pGPU6/GFP/Neo-sh710、sh1329、sh1436)和阴性对照质粒(pGPU6/GFP/Neo-shNC);脂质体介导法将重组质粒转染到NCI-H460细胞系中,并对稳定转染的细胞进行克隆,使用Western印迹和RT-PCR检测DAL-1基因的表达水平,并计算表达抑制率。MTT法检测转染后的NCI-H460增殖水平,Transwell小室检测侵袭和迁移能力,讨论细胞抑制DAL-1后的侵袭和迁移水平。以R语言为基础,以Bioconductor为编程环境,利用多种成熟的软件包设计了用于分析和挖掘基因芯片数据的系统。下载GEO中已经上传的Affymetrix寡核苷酸基因芯片,包含人类NSCLC的原始数据GSE33532,对该基因芯片的原始数据进行了包括数据下载、标准化处理、质控、差异基因的筛选与分析、GO分析、聚类以及通路总结,进一步分析基因调控网络以及分子相互作用。研究结果:1.DAL-1基因的RNAi沉默效应影响NCI-H460细胞设计和构建靶向DAL-1的pGPU6/GFP/Neo-DAL-1shRNA质粒和阴性对照质粒pGPU6/GFP/Neo-shNC,建立DAL-L稳定沉默的细胞NCI-H460-sh1329和阴性对照NCI-H460-shNC细胞。与阴性对照和空白质粒(NCI-H460/ET)相比,NCI-H460-sh1329组DAL-1细胞抑制率采用RT-PCR法检测达到88.17%,Western blot检测也得到了类似的结果。因此,认为sh1329有效的抑制了dal-1的表达,被称之为nci-h460-sh1329。nci-h460-shnc与nci-h460/et的dal-1表达抑制率并没有显著性的改变。2.抑制dal-1对nci-h460侵袭和迁移能力的影响采用mtt法检测细胞增殖能力,我们发现与阴性对照组nci-h460-shnc和空质粒转染组nci-h460/et相比,nci-h460-sh1329的增殖更快(p0.05)。采用基底膜侵袭实验检测细胞的侵袭和迁移能力,我们发现与阴性对照组nci-h460-shnc和空质粒转染nci-h460/et相比,nci-h460-sh1329细胞的侵袭和迁移显著增加(p0.05)。3.dal-1/4.1b的缺失或过表达改变了emt转移标记物的表达通过真核细胞转染shrna质粒的方法抑制dal-1/4.1b的表达后e-cadherin和β-catenin的表达下降,而vimentinb表达上升;反之,dal-1/4.1b的过表达则起到相反的效果。4.tgf-β诱导dal-1/4.1b的表达经tgf-β诱导后dal-1/4.1b的mrna及蛋白表达水平显著增加。5.nsclc生物信息学分析筛选到gse33532数据组的49个差异表达基因,其中多组基因都已经在实验中证实与nsclc存在密切相关的联系,其中血管平滑肌细胞收缩通路是nsclc的关键调节单位。结论:成功构建dal-1shrna表达质粒(pgpu6/gfp/neo-sh710、sh1329、sh1436)和阴性对照质粒(pgpu6/gfp/neo-shnc);建立了稳定的dal-1的沉默nci-h460-sh1329和阴性对照nci-h460-shnc细胞系。抑制dal-1后,nci-h460的增殖、迁移和侵袭水平升高,表明dal-1能抑制非小细胞肺癌迁移和侵袭。成功构建了生物信息学系统分析得到nsclc与正常肺组织细胞的差异基因以关键通路。本研究的创新性之处:1.靶向dal-1构建pgpu6/gfp/neo-dal-1shrna表达载体,自行设计sirna序列。实验结果说明sirna可以有效抑制nci-h460的dal-1表达水平,通过构建dal-1稳定抑制的细胞株,可以为探索dal-1基因在nsclc中的作用机理提供了合适的模式细胞;2.采用RNAi技术分析DAL-1在NCI-H460增殖、迁移和侵袭的功能水平,DAL-1与NSCLC转移侵袭等的关系并讨论其作用机理,为进一步探索NSCLC的产生与发展提供理论基础。3.利用生物信息学方法分析GEO中的GSE33532的基因数据,通过差异基因和通路分析找到关键的调节单位血管平滑肌细胞收缩通路,为今后进一步研究肺癌中分子作用机制提供基础。
[Abstract]:Background: at present, lung cancer is the highest incidence and death rate of malignant tumors in the world, of which Non-small cell lung cancer (NSCLC) accounts for the poor prognosis of 80%. in the late 80%. of all lung cancers. Invasion and metastasis are the main causes of death. Therefore, the molecular mechanism of NSCLC metastasis and invasion is studied and the related molecular targets are explored. The purpose of this study is to focus on the mechanism of the DAL-1 gene in the invasion and migration of NSCLC so as to provide a new target and theoretical basis for the clinical treatment of NSCLC. Research methods: 3 specific DAL-1 shRNA expression plasmids, based on the human DAL-mRNA coding sequence, are designed and incorporated into the target DAL-1. PGPU6/GFP/Neo-sh710, sh1329, sh1436) and negative control plasmids (pGPU6/GFP/Neo-shNC). The recombinant plasmid was transfected into the NCI-H460 cell line by liposome mediated method, and the stable transfected cells were cloned. The expression level of DAL-1 gene was detected by Western blot and RT-PCR, and the expression inhibition rate.MTT method was used to detect the NCI-H460 of the transfected NCI-H460. Proliferation level, Transwell cell detection of invasion and migration ability, discuss the invasion and migration level after cell inhibition of DAL-1. Based on R language and using Bioconductor as the programming environment, a variety of mature software packages are used to design a system for analyzing and mining gene chip data. Download the Affymetrix oligonucleotides that have been uploaded in GEO. Because of the chip, including the original data GSE33532 of human NSCLC, the original data of the gene chip were carried out including data downloading, standardization, quality control, screening and analysis of differential genes, GO analysis, clustering and pathway summary, further analysis of gene regulation network and molecular interaction. Research results: RNAi silencing of 1.DAL-1 gene. The effect affected NCI-H460 cells to design and construct the pGPU6/GFP/Neo-DAL-1shRNA plasmid and negative control plasmid pGPU6/GFP/Neo-shNC targeting DAL-1, to establish DAL-L stable silent cells NCI-H460-sh1329 and negative control NCI-H460-shNC cells. Compared with negative control and blank plasmid (NCI-H460/ET), the inhibition rate of DAL-1 cells in NCI-H460-sh1329 group The detection of 88.17% and Western blot detected by RT-PCR method also obtained similar results. Therefore, it is believed that sh1329 effectively inhibits the expression of DAL-1, which is called the DAL-1 expression inhibition rate of nci-h460-sh1329.nci-h460-shnc and nci-h460/et and has no significant change in the effect of.2. inhibition DAL-1 on NCI-H460 invasion and migration The proliferation ability of the cells was detected by the method. We found that the proliferation of nci-h460-sh1329 was faster than that of the negative control group nci-h460-shnc and the empty plasmid transfection group nci-h460/et (P0.05). The invasion and migration ability of the cells was detected by the basement membrane invasion test. We found that the NCI-H460 was compared with the negative control group, nci-h460-shnc and empty plasmid transfection nci-h460/et, NCI-H460. The invasion and migration of -sh1329 cells increased significantly (P0.05) the deletion or overexpression of.3.dal-1/4.1b changed the expression of EMT transfer markers by transfecting shRNA plasmids into the eukaryotic cells to inhibit the expression of dal-1/4.1b and the expression of E-cadherin and beta -catenin, while the expression of vimentinb increased; conversely, the overexpression of dal-1/4.1b was in phase. The inverse effect of.4.tgf- beta induced expression of dal-1/4.1b by tgf- beta induced mRNA and protein expression level of dal-1/4.1b significantly increased.5.nsclc bioinformatics analysis to screen 49 differentially expressed genes in the gse33532 data group, and many of these genes have proved to be closely related to NSCLC in the experiment, including vascular smooth muscle. The cell contraction pathway is the key regulating unit of NSCLC. Conclusion: the dal-1shrna expression plasmid (pgpu6/gfp/neo-sh710, sh1329, sh1436) and negative control plasmid (pgpu6/gfp/neo-shnc) were successfully constructed, and a stable DAL-1 silencing nci-h460-sh1329 and negative control nci-h460-shnc cell line were established. The proliferation, migration and invasion of NCI-H460 were inhibited after DAL-1. The level of DAL-1 can inhibit the migration and invasion of non-small cell lung cancer. It has successfully constructed a bioinformatics system to analyze the differential genes between NSCLC and normal lung tissue as a key pathway. The innovation of this study is: 1. target to DAL-1 to construct pgpu6/gfp/neo-dal-1shrna surface vector and design siRNA sequence. Experimental results say SiRNA can effectively inhibit the DAL-1 expression of NCI-H460. By constructing a DAL-1 stable cell line, it can provide a suitable model cell for exploring the mechanism of DAL-1 gene in NSCLC; 2. the function level of DAL-1 in NCI-H460 proliferation, migration and invasion, DAL-1 and NSCLC metastasis are analyzed by RNAi technique and the relationship between DAL-1 and NSCLC metastasis and so on The mechanism of its action is discussed to provide a theoretical basis for further exploration of the production and development of NSCLC..3. is used to analyze the gene data of GSE33532 in GEO by bioinformatics method, and the key regulatory unit vascular smooth muscle cell contraction pathway is found through differential gene and pathway analysis, which can further study the mechanism of molecular action in lung cancer in the future. For the foundation.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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