【作者】 杨旭东；

【导师】 杜贾军；

【作者基本信息】 山东大学 ， 临床医学（本硕连读）（专业学位）， 2018， 硕士

【摘要】 研究背景及目的肺癌是近年来世界范围内发病率不断上升,死亡率居高不下的恶性肿瘤,其病理分型有许多种,而肺腺癌(lung adenocarcinoma,LUAD)是其中发病率最高的一种,且在所有肺癌死亡病人占有较高的比例。目前靶向治疗已广泛应用于肺腺癌的治疗,并且针对部分病人已经取得了显著的疗效,但由于靶向治疗的局限性如耐药性的产生等严重影响了现有靶向药物的治疗效果,更多影响肺腺癌发生和预后的潜在治疗靶位基因需要我们去发现。许多研究已经证实钙黏素家族中某些基因表达的改变对许多肿瘤的生成有着深刻的影响,但其与肺腺癌的关系至今仍没有研究和阐明。研究方法本文通过应用一系列生物信息学相关的分析方法,力求找出钙黏素家族中基因差异表达与肺腺癌之间的关系。首先使用肿瘤基因图谱数据库(TheCancer Genome Atlas,TCGA)和基因表达综合数据库(Gene Expression Omnibus,GEO)找出钙黏素家族在肺腺癌和正常组织中表达有差异的基因。接着应用Kaplan-Meier Plotter数据库找出这些差异表达基因当中与预后相关的基因,并且这种差异表达将进一步使用山东大学附属省立医院病人的组织通过实施实时定量 PCR 实验(quantitative Real-time Polymerase Chain Reaction,qRT-PCR)来进行证实。接下来再使用基因富集分析方法(Gene Set Enrichment Analysis,GSEA)找出与个别差异表达基因相关的潜在信号表达通路,探索与肿瘤生成相关的部分机制。此外本次研究我们还使用了人类癌症DNA甲基化和基因表达数据库(Database of DNA Methylation and Gene Expression in Human Cancer,methHC)分析个别差异表达基因的甲基化水平及其与差异表达之间的相关性。最后我们还通过Ubibrowseir数据库分析了和与差异表达基因密切相关的泛素连接酶。研究结果最终,我们找到了在肺腺癌组织和肺正常组织中存在明显差异表达且和预后相关的六个钙黏素家族基因,它们的差异表达情况分别是cadherin 3(CDH3,p=1.32×10-14,log2FC=2.777),CDH5(p=1.27× 10-67,log2FC=-2.550),CDH19(p=0.003,log2FC=-2.210),fat cadherin 4(FAT4,p=5.62X×10-8,log2FC=-1.164),protocadherin9(PCDH9,p=1.60X 10-6,log2FC=-4.18)和 PCDH17(p=7.24 X1(T18,log2FC=-3.29)。其中5个基因的高表达和预后呈正相关,这5个基因分别为 CDH5(p=0.00055;HR 0.66,95%CI 0.52-0.83),CDH19(p=0.012;HR 0.73,95%CI 0.57-0.93),FAT4（p=1× 10-16;HR 0.34,95%CI 0.26-0.45),PCDH9(p=1.2×10-7;HR 0.53,95%CI 0.41-0.67)和 PCDH17(p=6.2×10r8;HR0.38,95%CI 0.26-0.55),而 CDH3(p=7.6×10-5,HR 1.59,95%Cl 1.26-2.01)的高表达与预后呈负相关。qRT-PCR的实验结果证实了相对于正常肺组织,CHD3在肺腺癌当中高表达,而CDH5,CDH19和PCDH17在肺腺癌当中低表达。此外,基因的甲基化普遍存在于这6个差异表达基因当中,PCDH17中S-shelf的甲基化(r=0.446),CDH5 中上游 200bp 的甲基化(r=0.363)和 PCDH9 中 5’UTR 的甲基化(0.224)和各自基因的差异表达情况相关。此外,由于目前文献鲜有报道对CDH19基因的相关肿瘤调控信号的研究,故此我们重点对CDH19进行了系统的GSEA分析,找出了与其在肿瘤中低表达密切相关的通路,前3条通路分别为核因子-κB(Nuclear Factor-κB,NF-κB)结合通路,连接酶活性正向调控通路和蛋白酶体通路。此外GSEA分析结果示CDH19的表达与其泛素化紧密相关,泛素化过程可能对钙黏素家族在肿瘤当中的差异表达起着重要的作用。通过Ubibrower数据库我们找出了属于这6个差异表达基因各自可能性最大的泛素连接酶,其中FAT4和连接酶神经前体细胞发育下调蛋白4抗体(Neuronally Expressed Developmentally Downregulated 4,NEDD4)的相关性最高(CS=0.866,SS<0.001)。结论以上所有的数据表明钙黏素家族中的基因包括CDH3,CDH5,CDH19,FAT4,PCDH9以及PCDH17在肺腺癌和肺正常组织中的差异表达和预后相关,其表达情况可作为肺腺癌预后评估的相关因素。此外基因的甲基化和PCDH17、CDH5和CDH3的差异表达之间具有相关性,泛素化可能与钙黏素家族中的某些基因如CDH19的表达密切相关。当然由于本次多项研究大多基于生信分析,相关的研究结果还需要临床和基础实验研究的进一步巩固和证实。更多还原

【Abstract】 BACKGROUND:In recent years,lung cancer has become a common malignant tumor with growing incidence rate and high mortality.This mortal cancer consists some histopathological forms,lung adenocarcinoma(LUAD)is one of the forms that with highest incidence rate and accounts for approximately 40%lung cancer death yet.Although targeted therapy has applied to the therapy of LUAD and acquired obvious effects in part of the patients,more potential genes that are related to the tumorigenesis and prognosis of LUAD are need to be found as the limitation of targeted therapy such as drug resistance.Genetic expression alterations of cadherin super-family members have been demonstrated to be important in tumorigenesis and progression of various cancers,however,the effects of most cadherins in LUAD were still unclear yet.METHODS:Here,based on bioinformatics analysis,we strove to elucidate the relationship between genes expression of cadherin family and LUAD.We first of all found out the differential expression genes(DEGs)between LUAD and lung normal tissues by The Cancer Genome Atlas(TCGA)and Gene expression Omnibus(GEO).And Kaplan-Meier Plotter database was applied to select the DEGs with significant prognosis.The DEGs would be assured by the patients’ tissues in Shandong Provincial hospital.Then Gene set enrichment analysis(GSEA)was conducted to find out potential signaling pathways of these DEGs.Finally,Database of DNA Methylation and Gene Expression in Human Cancer(methHC)and Ubibrower were used to elucidate the methylation level of DEGs and ubiquitin ligases(E3s)of DEGs that related with tumorigenesis respectively.RESULTS:Ultimately,we found six DEGs including cadherin 3(CDH3,p=1.32×10-14,log2FC=2.777),CDH5(p=1.27× 10-67,log2FC=-2.550),CDH19(p=0.003,log2FC=-2.210),fat cadherin 4(FAT4,p=5.62×10-8,log2FC=-1.164),protocadherin 9(PCDH9,p=1.60 ×10-6,log2FC=-4.18)and PCDH17(p=7.24 × 10-18,log2FC=-3.29).Among them,high expression of CDH5(p=0.00055;HR 0.66,95%CI 0.52-0.83),CDH19(p=0.012;HR 0.73,95%C10.57-0.93),FAT4(p=1 × 10-16;HR 0.34,95%CI 0.26-0.45),PCDH9(p=1.2×10-7;HR 0.53,95%CI 0.41-0.67)and PCDH17(p=6.2×10-8;HR0.38,95%CI 0.26-0.55)were positively related to prognosis of LUAD,while high expression of CDH3(p=7.6×10-5,HR 1.59,95%Cl 1.26-2.01)was negatively related to prognosis of LUAD.Besides,the differential expression of CDH3,CDH5,CDH19,and PCDH17 were validated by qRT-PCR.Genes methylation was ubiquitous in these six DEGs,s-shelf methylation of PCDH17,(r= 0.446),upstream 200bp of CDH5(r=0.363)s and 5’UTR methylation of PCDH9(r=0.224)were correlated with their expression.As differential expression of CDH19 was validated by our qRT-PCR and no research aiming at CDH19 reported in LUAD yet,it became the gene of great interest for GSEA analysis.The top 3 enrichment pathways with low expression of CDH19 including NF-κB binding,positive regulation of ligase activity and proteasome complex.Since ubiquitination was Also elucidated in GSEA analysis result of CDH19,it raised the possibility that degradation of CDH19 even other DEGs were related with E3s.Based on this,we also found out the possible E3s of the six DEGs,and the E3 neuronally expressed developmentally downregulated 4(NEDD4)of FAT4 had the highest confidence score(CS=0.866,SS<0.001).CONCUSIONS:These data suggested that the differential expression of CDH3,CDH5,CDH19,FAT4,PCDH9,and PCDH17 between LUAD and normal lung tissues were correlated with prognosis,and their differential expression levels can be considered as the prognosis indicators.Besides,methylation of PCDH17,CDH5 and CDH3 was related to their expression levels.Moreover,ubiquitination may strongly associate with expression levels of some cadherin super-family members such as CDH19.As most of the study results were based on bio information analysis,more definitive basic and clinical experiments should be conducted to validate these analysis results.更多还原