【作者】 余锋；

【导师】 熊建萍；

【作者基本信息】 南昌大学 ， 肿瘤学（专业学位）， 2016， 博士

【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(p<0.05)。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(p<0.05)。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。更多还原

【Abstract】 Background:Lung cancer is currently the world’s highest incidence and mortality of malignant tumors, including Non-small cell lung cancer(NSCLC). Invasion and metastasis are the main causes of death, so the research on the molecular mechanism of NSCLC invasion and metastasis is the key to be performed. Objective:This study focused on the role of DAL-1 gene in NSCLC cell migration and invasion, the possible mechanism of the gene in NSCLC, the main cause of inactivation and its possible therapeutic approach. We look forward to finding a new target for clinical treatment of NSCLC and providing experimental basis. Methods:According to the sequence of human DAL-mRNA, 3 specific shRNA DAL-1 expressionplasmids(pGPU6/GFP/Neo-sh710, sh1436, sh1329) and negative control plasmid(shNC) were designed and synthesized. The NCI-H460 cell line was transfected with recombinant plasmid, and the expression of DAL-1 gene was detected by Western blot and RT-PCR, respectively. MTT assay was used to detect the DAL-1 gene silencing cell proliferation activity. Transwell chamber was used to detect cell migration and invasion ability of NSCLC cell.Based on the open source R language programming platform, Bioconductor was used as a biological computing environment, and a variety of software packages were used to construct a data analysis and data mining system for microarray data. The original data GSE33532 of Affymetrix oligonucleotide microarray was published in the public database GEO. The data was analyzed, including the acquisition, pretreatment, quality detection, quality detection, differential expression gene screening, GO analysis, pathway analysis, gene expression regulatory network, molecular interaction network analysis and visualization. Results:1. The effect of DAL-1 gene on NCI-H460 cells.The pGPU6/GFP/Neo-DAL-1sh RNA plasmid and the negative control plasmid pGPU6/GFP/Neo-shNC were constructed, and the NCI-H460-sh1329 cell lines with stable silencing of DAL-l were established. Compared with negative and empty plasmid control, the DAL-l inhibition rate of sh1329 transfected cells was 88%, and the Western blot assay was got similar results. There was no significant difference between in NCI-H460-shNC and NCI-H460/ET transfection groups.2. The effect of silencing DAL-1 gene on the biological behavior of NCI-H460 cells.Compared to the negative control group(NCI-H460-shNC) and NCI-H460/ET cells, NCI-H460-sh1329 had more invasion rate with significant difference(P<0.05).3. The deletion or overexpression of DAL-1/4.1B changed the expression of EMT markers.Silencing of DAL-1/4.1B RNA changed the expression of EMT markers, including E-cadherin andβ-catenin, and the overexpression of DAL-1/4.1B had the opposite effect.4. TGF-β induced DAL-1/4.1B expression.The expression of DAL-1/4.1B at protein and mRNA level was increased after TGF-β induction. 4.1B DAL-1/ deficient cells were weaken on EMT, migration and invasion induced by TGF-β.5. Bioinformation of NSCLC genome in GSE3353249 Differential expression genes were selected from the GSE33532 data set, and the vascular endothelial contration pathway was the key regulatory units of NSCLC. Conclusion:The shRNA pGPU6/GFP/Neo-DAL-1 plasmid and the negative control plasmid pGPU6/GFP/Neo-shNC were constructed successfully, and the NCI-H460-sh1329 cell lines with stable silencing DAL-1 were established. After silencing of DAL-1 gene, the proliferation, migration and invasion of NCI-H460 cells were increased, which indicated that DAL-1 could inhibit the proliferation, migration and invasion of NSCLC cells. Vascular endothelial contraction pathway might regulate NSCLC. Innovations:1. DAL-1 siRNA sequences were designed to construct pGPU6/GFP /Neo-DAL-1 shRNA expression vector. The DAL-1 expression of NCI-H460 cells could be inhibited effectively by pGPU6/GFP/Neo-DAL-1 NCI-H460, and the DAL-1 inhibition cells line was established to further study;2. RNAi technique is used to analyze the effect of DAL-1 on the proliferation, migration and invasion of NCI-H460 cells, and the relationship between DAL-1 and NSCLC is discussed, which provides an experimental basis for further study of the development on non small cell lung cancer.3. Using bioinformatics method to analyze the gene data of GSE33532, and KEGG were used to get the key regulatory units which contain vascular endothelial contraction pathway, providing a basis for further study on the molecular mechanism of NSCLC.更多还原