转录组数据分析肺腺癌和肺鳞状细胞癌中基因的差异表达
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摘要
目的对非小细胞肺癌两种亚型肺腺癌和肺鳞状细胞癌的基因差异表达进行生物信息学分析,寻找非小细胞肺癌潜在的生物学标志物运用于临床诊断,进而提高患者生存率。方法获取肺腺癌和肺鳞状细胞癌的基因表达谱分析数据集,使用R语言处理,t检验分析鉴定肺腺癌和肺鳞状细胞癌之间的转录组差异,并通过维恩图显示所鉴定的基因差异表达。从GEO获得差异表达的基因,用DAVID及IPA进行分析,进一步确定潜在可用于鉴别肺腺癌和肺鳞状细胞癌的几种信号通路和生物标记。利用TCGA数据和临床肺癌样本中的生物标记表达,验证Osteoarthritis pathway和LXR/RXR分别在肺腺癌和肺鳞状细胞癌的基因差异表达;挑选miR-181b-5p及其靶基因WNT5A和MBD2进行验证,收集23例肺鳞癌患者的肺肿瘤组织(23例肿瘤组织,实验组)及其邻近肺组织(23例癌旁肺组织,对照组),检测miR-181b-5p和其靶基因WNT5A和MBD2的表达差异用于鉴别肺腺癌和肺鳞状细胞癌。结果 GEO数据分析结果揭示肺腺癌和肺鳞状细胞癌的差异表达基因:肺腺癌中有851个DEGs(276个上调,575个下调),而肺鳞状细胞癌中有885个DEGs(406个上调,479个下调)。DAVID和IPA分析差异表达基因结果显示,白细胞迁移和炎症反应在肺腺癌中比肺鳞状细胞癌富集程度更高,Osteoarthritis pathway在肺腺癌中是抑制状态,而在肺鳞状细胞癌中显示是激活状态。IPA对重要转录因子、细胞因子和miRNA的分析结果显示肺腺癌和肺鳞状细胞癌之间的区别主要是:转录因子(GATA4,RELA,YBX1,TP63和MBD2);细胞因子(WNT5A和IL-1A)和microRNA(miR-34a,miR-181b和miR-15a)。其中miR-34a和IL-1A、miR-15a和YBX1、miR-181b和WNT5A和MBD2可作为鉴别非小细胞肺癌亚型的成对microRNA和mRNA靶点。临床样本实验结果支持miR-181b-5p的表达上升和WNT5A的表达下调可视为诊断肺鳞状细胞癌的分子标记物。结论通过对转录组数据分析,发现了肺腺癌和肺鳞状细胞癌的候选基因、成对的microRNA鉴别肺腺癌和肺鳞状细胞癌,为其鉴别诊断和治疗提供了新的思路。
Objective To analyze the differentially expressed genes(DEGs) between lung adenocarcinoma(LUAD) and lung squamous cell carcinoma(LUSC) with bioinformatics analysis and search for potential biomarkers for clinical diagnosis of nonsmall cell lung cancer(NSCLC). Methods The gene expression profiling datasets of LUAD and LUSC were acquired. The transcriptome differences between LUAD and LUSC were identified using R language processing and t-test analysis. The differential expressions of the genes were shown by Venn diagram. The DEGs identified by GEO2 R were analyzed with DAVID and Ingenuity Pathway Analysis(IPA) to identify the signaling pathways and biomarkers that could be used for differential diagnosis of LUAD and LUSC. The TCGA data and the biomarker expression data from clinical lung cancer samples were used to verify the differential expressions of the Osteoarthritis pathway and LXR/RXR between LUAD and LUSC. We further examined the differential expressions of miR-181 and its two target genes, WNT5 A and MBD2, in 23 clinical specimens of lung squamous cell carcinoma and the paired adjacent tissues. Results GEO data analysis identified 851 DEGs(including 276 up-regulated and 575 down-regulated genes) in LUAD and 885 DEGs(including 406 up-regulated and 479 down-regulated genes) in LUSC. DAVID and IPA analysis revealed that leukocyte migration and inflammatory responses were more abundant in LUAD than in LUSC. Osteoarthritis pathway was inhibited in LUAD and activated in LUSC. IPA analysis showed that transcription factors(GATA4, RELA, YBX1, TP63 and MBD2), cytokines(WNT5 A and IL1 A) and microRNAs(miR-34 a, miR-181 b and miR-15 a) differed significantly between LUAD and LUSC. miR-34 a with IL-1 A, miR-15 a with YBX1,and miR-181 b with WNT5 A and MBD2 could serve as the paired microRNA and mRNA targets for differential diagnosis of NSCLC subtypes. Analysis of the clinical samples showed an increased expression of miR-181 b-5 p and the down-regulation of WNT5 A, which could be used as molecular markers for the diagnosis of LUSC. Conclusion Through transcriptome analysis, we identified candidate genes, paired microRNAs and pathways for differentiating LUAD and LUSC, and they can provide novel differential diagnosis and therapeutic strategies for LUAD and LUSC.
Objective To analyze the differentially expressed genes(DEGs) between lung adenocarcinoma(LUAD) and lung squamous cell carcinoma(LUSC) with bioinformatics analysis and search for potential biomarkers for clinical diagnosis of nonsmall cell lung cancer(NSCLC). Methods The gene expression profiling datasets of LUAD and LUSC were acquired. The transcriptome differences between LUAD and LUSC were identified using R language processing and t-test analysis. The differential expressions of the genes were shown by Venn diagram. The DEGs identified by GEO2 R were analyzed with DAVID and Ingenuity Pathway Analysis(IPA) to identify the signaling pathways and biomarkers that could be used for differential diagnosis of LUAD and LUSC. The TCGA data and the biomarker expression data from clinical lung cancer samples were used to verify the differential expressions of the Osteoarthritis pathway and LXR/RXR between LUAD and LUSC. We further examined the differential expressions of miR-181 and its two target genes, WNT5 A and MBD2, in 23 clinical specimens of lung squamous cell carcinoma and the paired adjacent tissues. Results GEO data analysis identified 851 DEGs(including 276 up-regulated and 575 down-regulated genes) in LUAD and 885 DEGs(including 406 up-regulated and 479 down-regulated genes) in LUSC. DAVID and IPA analysis revealed that leukocyte migration and inflammatory responses were more abundant in LUAD than in LUSC. Osteoarthritis pathway was inhibited in LUAD and activated in LUSC. IPA analysis showed that transcription factors(GATA4, RELA, YBX1, TP63 and MBD2), cytokines(WNT5 A and IL1 A) and microRNAs(miR-34 a, miR-181 b and miR-15 a) differed significantly between LUAD and LUSC. miR-34 a with IL-1 A, miR-15 a with YBX1,and miR-181 b with WNT5 A and MBD2 could serve as the paired microRNA and mRNA targets for differential diagnosis of NSCLC subtypes. Analysis of the clinical samples showed an increased expression of miR-181 b-5 p and the down-regulation of WNT5 A, which could be used as molecular markers for the diagnosis of LUSC. Conclusion Through transcriptome analysis, we identified candidate genes, paired microRNAs and pathways for differentiating LUAD and LUSC, and they can provide novel differential diagnosis and therapeutic strategies for LUAD and LUSC.
引文
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[31]Liu C,Zhou Y,Li M,et al.Absence of GdX/UBL4A protects against inflammatory diseases by regulating NF-small ka,CyrillicBsignaling in macrophages and dendritic cells[J].Theranostics,2019,9(5):1369-84.
[32]Jagannathan L,Jose CC,Arita AA,et al.Nuclear factor kappa B1/RelA mediates inflammation in human lung epithelial cells at atmospheric oxygen levels[J].J Cell Physiol,2016,231(7):1611-20.
[33]Chen YJ,Guo YN,Shi K,et al.Down-regulation of microRNA-144-3p and its clinical value in non-small cell lung cancer:a comprehensive analysis based on microarray,miRNA-sequencing,and quantitative real-time PCR data[J].Respir Res,2019,20(7):48-59.
[34]Li G,Wang K,Wang J,et al.miR-497-5p inhibits tumor cell growth and invasion by targeting SOX5 in non-small-cell lung cancer[J].JCell Biochem,2019,283(12):1570-83.
[35]Zhuangandy B,Cheng M.MicroRNA629 inhibition suppresses the viability and invasion of nonsmall cell lung cancer cells by directly targeting RUNX3[J].Mol Med Rep,2019,11(4):315-26.
[36]Wang H.Predicting MicroRNA biomarkers for cancer using phylogenetic tree and microarray analysis[J].Int J Mol Sci,2016,17(5):257-68.
[37]Gyoba J,Shan S,Roa W,et al.Diagnosing lung cancers through examination of Micro-RNA biomarkers in blood,plasma,serum and sputum:a review and summary of current literature[J].Int J Mol Sci,2016,17(4):494-508.
[2]Lim SL,Jia ZA,Lu YH,et al.Metabolic signatures of four major histological types of lung cancer cells[J].Metabolomics,2018,14(9):118-27.
[3]Ettinger DS,Akerley W,Borghaei H,et al.Non-small cell lung cancer,version 2.2013[J].J Natl Compr Canc Netw,2013,11(6):645-53.
[4]Bjorkqvist AM,Husgafvel-Pursiainen K,Anttila S,et al.DNA gains in 3q occur frequently in squamous cell carcinoma of the lung,but not in adenocarcinoma[J].Genes Chromosomes Cancer,1998,22(1):79-82.
[5]Cooper WA,Lam DC,O'toole SA,et al.Molecular biology of lung cancer[J].J Thorac Dis,2013,5(5):S479-90.
[6]Ceppi P,Volante M,Saviozzi S,et al.Squamous cell carcinoma of the lung compared with other histotypes shows higher messenger RNAand protein levels for thymidylate synthase[J].Cancer,2006,107(7):1589-96.
[7]Davidson MR,Gazdarandb AF.E.clarke.the pivotal role of pathology in the management of lung cancer[J].J Thorac Dis,2013,37(5):463-78.
[8]Langer CJ,Besse B,Gualberto A,et al.The evolving role of histology in the management of advanced non-small-cell lung cancer[J].J Clin Oncol,2010,28(36):5311-20.
[9]Tomasini P,Khobta N,Greillier LA.Ipilimumab:its potential in non-small cell lung cancer[J].Ther Adv Med Oncol,2012,4(2):43-50.
[10]Paez JG,J?nne PA,Lee JC,et al.EGFR mutations in lung cancer:correlation with clinical response to gefitinib therapy[J].Science,2004,304(5676):1497-500.
[11]MathéEA,Nguyen GH,Bowman ED,et al.MicroRNA expression in squamous cell carcinoma and adenocarcinoma of the esophagus:associations with survival[J].Clin Cancer Res,2009,15(19):6192-200.
[12]Patnaik S,Mallick R,Kannisto E,et al.MiR-205 and MiR-375MicroRNA assays to distinguish squamous cell carcinoma from adenocarcinoma in lung cancer biopsies[J].J Thorac Oncol,2015,10(3):446-53.
[13]da WH,Shermanandr BT,Lempicki A.Systematic and integrative analysis of large gene lists using David bioinformatics resources[J].Nat Protoc,2009,4(1):44-57.
[14]Li JH,Liu S,Zhou H,et al.Starbase v2.0:decoding miRNA-ceRNA,miRNA-ncRNA and protein-RNA interaction networks from large-scale CLIP-Seq data[J].Nucleic Acids Res,2014,42(Database issue):D92-7.
[15]Li Y,Gu J,Xu FK,et al.Transcriptomic and functional network features of lung squamous cell carcinoma through integrative analysis of GEO and TCGA data[J].Sci Rep,2018,8(1):15834-45.
[16]Xie LX,Dang YF,Guo JS,et al.High KRT8 expression independently predicts poor prognosis for lung adenocarcinoma patients[J].Genes(Basel),2019,10(1):1279-92.
[17]Zhao ZX,Liu BJ,Sun J,et al.Baicalein inhibits orthotopic human Non-Small cell lung cancer xenografts via Src/Id1 pathway[J].Evid Based Complement Alternat Med,2019,98(6):162-73.
[18]庄铭锴,陈丰霖.上皮间质转化相关的生物标志在胃癌中的研究进展[J].世界华人消化杂志,2013,21(30):3204-10.
[19]Liu SY,Wang XJ,Qin WY,et al.Transcription factors contribute to differential expression in cellular pathways in lung adenocarcinoma and lung squamous cell carcinoma[J].Interdiscip Sci,2018,10(4):836-47.
[20]Li Y,Sun N,Lu ZL,et al.Prognostic alternative mRNA splicing signature in non-small cell lung cancer[J].Cancer Lett,2017,393(1):40-51.
[21]Dai YB,Miao YF,Wu WF,et al.Ablation of liver X receptors alpha and beta leads to spontaneous peripheral squamous cell lung cancer in mice[J].Proc Natl Acad Sci USA,2016,113(27):7614-9.
[22]Yang Z,Zhuan B,Yan Y,et al.Integrated analyses of copy number variations and gene differential expression in lung squamous-cell carcinoma[J].Biol Res,2015,48(7):108-16.
[23]Zhang S,Li MF,Ji HB,et al.Landscape of transcriptional deregulation in lung cncer[J].BMC Genomics,2018,19(1):435-49.
[24]Shi YX,Wang Y,Li X,et al.Genome-wide DNA methylation profiling reveals novel epigenetic signatures in squamous cell lung cancer[J].BMC Genomics,2017,18(11):901-15.
[25]Cheng H,Yang XP,Si H,et al.Genomic and transcriptomic characterization links cell lines with aggressive head and neck cancers[J].Cell Rep,2018,25(5):1332-43.
[26]Yoshida M,Yokota E,Sakuma T,et al.Development of an integrated CRISPRi targeting DeltaNp63 for treatment of squamous cell carcinoma[J].Oncotarget,2018,9(49):29220-32.
[27]Eun YG,Lee D,Lee YC,et al.Clinical significance of YAP1activation in head and neck squamous cell carcinoma[J].Oncotarget,2017,8(67):111130-43.
[28]Mishra A,Bharti AC,Varghese P,et al.Differential expression and activation of NF-kappa B family proteins during oral carcinogenesis:role of high risk human papillomavirus infection[J].Int J Cancer,2006,119(12):2840-50.
[29]Taggart CC,Cryan SA,Weldon S,et al.Secretory leucoprotease inhibitor binds to NF-kappa B binding sites in monocytes and inhibits p65 binding[J].J Exp Med,2005,202(12):1659-68.
[30]Huang X,Guo B,Liu S,et al.Neutralizing negative epigenetic regulation by HDAC5 enhances human haematopoietic stem cell homing and engraftment[J].Nat Commun,2018,19(21):2741-56.
[31]Liu C,Zhou Y,Li M,et al.Absence of GdX/UBL4A protects against inflammatory diseases by regulating NF-small ka,CyrillicBsignaling in macrophages and dendritic cells[J].Theranostics,2019,9(5):1369-84.
[32]Jagannathan L,Jose CC,Arita AA,et al.Nuclear factor kappa B1/RelA mediates inflammation in human lung epithelial cells at atmospheric oxygen levels[J].J Cell Physiol,2016,231(7):1611-20.
[33]Chen YJ,Guo YN,Shi K,et al.Down-regulation of microRNA-144-3p and its clinical value in non-small cell lung cancer:a comprehensive analysis based on microarray,miRNA-sequencing,and quantitative real-time PCR data[J].Respir Res,2019,20(7):48-59.
[34]Li G,Wang K,Wang J,et al.miR-497-5p inhibits tumor cell growth and invasion by targeting SOX5 in non-small-cell lung cancer[J].JCell Biochem,2019,283(12):1570-83.
[35]Zhuangandy B,Cheng M.MicroRNA629 inhibition suppresses the viability and invasion of nonsmall cell lung cancer cells by directly targeting RUNX3[J].Mol Med Rep,2019,11(4):315-26.
[36]Wang H.Predicting MicroRNA biomarkers for cancer using phylogenetic tree and microarray analysis[J].Int J Mol Sci,2016,17(5):257-68.
[37]Gyoba J,Shan S,Roa W,et al.Diagnosing lung cancers through examination of Micro-RNA biomarkers in blood,plasma,serum and sputum:a review and summary of current literature[J].Int J Mol Sci,2016,17(4):494-508.