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膀胱癌(BC)在泌尿系统侵袭性恶性肿瘤中的发生率最高,也是全球最常见的癌症之一[1]。全球每年约有165 000人因BC死亡,其生物学行为复杂,且极易复发和转移[2-3]。目前,BC的诊断和治疗仍存在很大的不足,其治疗方法和预后与是否存在肌层浸润有关。非肌肉浸润型BC(NMIBC)预后较好,而肌肉浸润型(MIBC)恶性程度很高[4]。对于后者,根治性膀胱切除加盆腔淋巴结清扫是标准治疗方案,但预后较差[3-5]。因此,迫切需要新的途径为BC的预后和治疗提供信息。本研究利用TCGA数据库分析BC的mRNA表达水平,从中筛选出差异表达的基因。并对差异基因进行系统性分析,为寻找更有效的分子治疗方法提供参考。
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通过对TCGA数据库中414个BLCA样本和19个对应的对应的癌旁组织样本处理分析,得到1 538个差异表达基因,其中有1 088个为上调,450个下调。利用R软件绘制差异基因的火山图(见图 1)。
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应用数据库DAVID进行GO富集分析评估了差异基因的潜在生物学功能。发现差异基因在生物过程(biological process,BP)方面主要富集在:rDNA上的染色质沉默、DNA复制依赖的核小体组装、蛋白异构化、核小体组装、肌肉收缩、细胞蛋白代谢过程、端粒组织、肌丝滑动、女性妊娠等;在细胞组分(CC)方面主要富集在:细胞外间隙、核小体、Z盘、蛋白类细胞外基质、胞外基质、中间丝、胞外外泌体等;在分子功能(MF)方面主要富集在:结构分子活性、序列特异性结合、蛋白异二聚化活性等(见图 2与表 1)。
类别 名称 n 百分率/% P FDR MF 结构分子活性 40 3.57 < 0.01 < 0.01 MF 序列特异性DNA结合 55 4.92 < 0.01 < 0.01 MF 蛋白质异二聚活性 51 4.56 < 0.01 < 0.01 MF 肌肉的结构成分 13 1.16 < 0.01 < 0.01 MF 肝素结合 24 2.14 < 0.01 < 0.01 CC 胞外区 182 16.26 < 0.01 < 0.01 CC 细胞外间隙 146 13.05 < 0.01 < 0.01 CC 核小体 32 2.86 < 0.01 < 0.01 CC Z盘 26 2.32 < 0.01 < 0.01 CC 核小体 15 1.34 < 0.01 < 0.01 CC 蛋白质细胞外基质 36 3.22 < 0.01 < 0.01 CC 细胞外基质 37 3.31 < 0.01 < 0.01 CC 中间丝 21 1.88 < 0.01 < 0.01 CC 胞外外泌体 179 16.00 < 0.01 < 0.01 BP 肌肉收缩 25 2.23 < 0.01 < 0.01 BP 核小体组装 26 2.32 < 0.01 < 0.01 BP 细胞蛋白质代谢过程 24 2.14 < 0.01 < 0.01 BP 端粒组织 12 1.07 < 0.01 < 0.01 BP 蛋白质异四聚化 14 1.25 < 0.01 < 0.01 BP DNA复制依赖性核小体组装 12 1.07 < 0.01 < 0.01 BP rDNA染色质沉默 12 1.07 < 0.01 < 0.01 BP 肌丝滑动 12 1.07 < 0.01 < 0.01 BP 女性怀孕 17 1.52 < 0.01 < 0.01 表 1 差异表达基因的GO富集分析结果
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应用数据库DAVID进行KEGG富集分析检查了差异基因涉及的潜在信号通路。发现差异基因主要涉及癌症中的转录失调、酪氨酸代谢、cGMP-PKG信号通路、系统性红斑狼疮、酒精中毒、扩张型心肌病、肥厚型心肌病、神经活性配体-受体相互作用等(见图 3和表 2)。
名称 n 百分率/% P 系统性红斑狼疮 33 2.95 < 0.01 酗酒 36 3.22 < 0.01 扩张型心肌病 17 1.52 < 0.01 肥厚型心肌病(HCM) 16 1.43 < 0.01 神经活性配体-受体相互作用 27 2.41 < 0.01 病毒致癌作用 22 1.97 < 0.01 胰岛素分泌 13 1.16 < 0.01 钙信号通路 20 1.79 < 0.01 心肌细胞中的肾上腺素能信号 17 1.52 < 0.01 血管平滑肌收缩 15 1.34 < 0.01 致心律失常性右心室心肌病(ARVC) 11 0.98 < 0.01 年轻人的成年型糖尿病 7 0.63 < 0.01 唾液分泌 12 1.07 < 0.01 心肌收缩 11 0.98 < 0.01 昼夜节律 12 1.07 < 0.01 胆碱能突触 13 1.16 < 0.01 催产素信号通路 15 1.34 < 0.01 视黄醇代谢 9 0.80 < 0.01 胰腺分泌物 11 0.98 < 0.01 药物代谢-细胞色素P450 9 0.80 < 0.01 cGMP-PKG信号通路 15 1.34 < 0.05 蛋白质消化吸收 10 0.89 < 0.05 酪氨酸代谢 6 0.54 < 0.05 Ⅱ型糖尿病 7 0.63 < 0.05 苯丙胺成瘾 8 0.71 < 0.05 补体和凝血级联 8 0.71 < 0.05 癌症中的转录失调 14 1.25 < 0.05 胃酸分泌 8 0.71 < 0.05 肌动蛋白细胞骨架的调节 16 1.43 < 0.05 表 2 差异表达基因的KEGG富集分析结果
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为分析差异表达基因之间相互作用关系,利用在线数据库STRING11.0对1 538个差异表达基因进行PPI网络构建,将相互作用分数设置为0.7,得到了相互作用网络图(见图 4)。
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利用Cytoscape软件插件Cytohubba,采用Betweenness算法得出INS、ACTN2、ALB、KNG1、白细胞介素-6(IL-6)、IGF2、FOS、间皮素(MSLN)、DMD、APCS为PPI网络中的前10的关键节点基因(见图 4),这10个关键基因中,INS、ALB、KNG1、IGF2、MSLN、APCS表达均为上调,ACTN2、IL-6、FOS、DMD下调(见图 5)。
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利用UALCAN数据库验证了10个关键基因在BC中的表达水平,结果显示,ACTN2、IL-6、FOS、DMD在BC中异常低表达(见图 6A),而ALB、MSLN的表达高于正常膀胱组织(见图 6B)。
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为了进一步探讨其在BC中的特异性表达。通过GEPIA数据库再次验证,分析结果与UALCAN数据库验证吻合,ACTN2、IL-6、FOS、DMD在BC中异常低表达(见图 7A),而ALB、MSLN在BC中高表达(见图 7B)。
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应用在线工具Kaplan Meier Plotter(http://kmplot.com/analysis/)对ACTN2、IL-6、FOS、DMD、ALB、MSLN等关键基因进行预后价值分析,发现基因IL-6、MSLN的表达情况与BC预后相关有统计学意义,在BC中IL-6表达越高预后越好(HR=0.46,P < 0.05),MSLN表达越高预后越差(HR=2.27,P < 0.05)(见图 8)。
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应用R软件分析IL-6与MSLN mRNA表达与BC不同临床及病理参数的关系显示, IL-6 mRNA的表达与病人年龄、肿瘤生长部位及和病理分期T分期有关而与其他参数无关(见图 9):55岁以上的BC病人的IL-6 mRNA表达水平高于低年龄组(P < 0.05);在BC病人病理分期T分期中,T1期病人的IL-6 mRNA表达水平低于于其他组(P < 0.05);肿瘤位于膀胱后壁组的BC病人IL-6 mRNA表达水平高于位于膀胱侧壁组(P < 0.05)。MSLN mRNA的表达与病人肿瘤生长部位及和病理分期N分期有关而与其他参数无关(见图 9):在BC病人病理分期N分期中,T3期病人的MSLN mRNA表达水平低于N0(P < 0.05)、N2组(P < 0.05);肿瘤位于膀胱前壁组的BC病人IL-6 mRNA表达水平高于位于膀胱侧壁组(P < 0.05)。
基于TCGA数据库探究膀胱癌关键基因的表达及预后作用
Expression and prognostic value of key genes in bladder cancer based on TCGA database
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摘要:
目的探究膀胱癌(BC)中基因的差异化表达及其预后作用。 方法从TCGA数据库中下载BC的基因表达数据,利用R软件筛选出肿瘤与正常组织间差异表达基因,并进行富集分析,评估其潜在生物学功能及信号通路。利用在线数据库STRING11.0构建了编码蛋白相互作用网络,进一步筛选关键基因。通过UALCAN、GEPIA数据库验证关键基因在BLCA中的表达水平。随后利用在线工具Kaplan Meier Plotter分析他们的预后情况。 结果得到1 538个基因存在差异表达,其中有1 088个上调,450个下调,富集分析发现差异基因主要与信号转导、物质代谢、免疫反应等信号通路相关。10个关键基因中,在BC中白细胞介素-6表达越高预后越好(HR=0.46,P < 0.05),间皮素表达越高预后越差(HR=2.27,P < 0.05)。 结论BC中存在众多差异表达的基因,他们对疾病进展存在影响,发现并利用他们在BC个体化治疗存在重要意义。 Abstract:ObjectiveTo explore the differential expressions and the prognostic role of genes in bladder cancer. MethodsThe gene expression data of bladder cancer were downloaded from TCGA database. The differentially expressed genes between tumor and normal tissues were screened by R software, and their potential biological functions and signal pathways were evaluated by enrichment analysis. A protein-coding interaction network was constructed using the online database STRING11.0 to further screen key genes. The expressions of key genes in bladder cancer was verified by GEPIA and UALCAN database. Then the online tool Kaplan Meier Plotter was used to analyze the prognosis. ResultsA total of 1 538 genes were differentially expressed, including 1 088 up-regulated and 450 down-regulated. Enrichment analysis showed that the differential genes were mainly related to signal transduction, substance metabolism, immune responses and other signal pathways. Among the 10 key genes screened, 2 genes have the prognostic value. ConclusionsThere are many differentially expressed genes in bladder cancer which have an impact on the progression of the disease. It is of great significance to find and use them in the individualized treatment of bladder cancer. -
Key words:
- bladder neoplasms /
- TCGA database /
- genes /
- differential expressions
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表 1 差异表达基因的GO富集分析结果
类别 名称 n 百分率/% P FDR MF 结构分子活性 40 3.57 < 0.01 < 0.01 MF 序列特异性DNA结合 55 4.92 < 0.01 < 0.01 MF 蛋白质异二聚活性 51 4.56 < 0.01 < 0.01 MF 肌肉的结构成分 13 1.16 < 0.01 < 0.01 MF 肝素结合 24 2.14 < 0.01 < 0.01 CC 胞外区 182 16.26 < 0.01 < 0.01 CC 细胞外间隙 146 13.05 < 0.01 < 0.01 CC 核小体 32 2.86 < 0.01 < 0.01 CC Z盘 26 2.32 < 0.01 < 0.01 CC 核小体 15 1.34 < 0.01 < 0.01 CC 蛋白质细胞外基质 36 3.22 < 0.01 < 0.01 CC 细胞外基质 37 3.31 < 0.01 < 0.01 CC 中间丝 21 1.88 < 0.01 < 0.01 CC 胞外外泌体 179 16.00 < 0.01 < 0.01 BP 肌肉收缩 25 2.23 < 0.01 < 0.01 BP 核小体组装 26 2.32 < 0.01 < 0.01 BP 细胞蛋白质代谢过程 24 2.14 < 0.01 < 0.01 BP 端粒组织 12 1.07 < 0.01 < 0.01 BP 蛋白质异四聚化 14 1.25 < 0.01 < 0.01 BP DNA复制依赖性核小体组装 12 1.07 < 0.01 < 0.01 BP rDNA染色质沉默 12 1.07 < 0.01 < 0.01 BP 肌丝滑动 12 1.07 < 0.01 < 0.01 BP 女性怀孕 17 1.52 < 0.01 < 0.01 表 2 差异表达基因的KEGG富集分析结果
名称 n 百分率/% P 系统性红斑狼疮 33 2.95 < 0.01 酗酒 36 3.22 < 0.01 扩张型心肌病 17 1.52 < 0.01 肥厚型心肌病(HCM) 16 1.43 < 0.01 神经活性配体-受体相互作用 27 2.41 < 0.01 病毒致癌作用 22 1.97 < 0.01 胰岛素分泌 13 1.16 < 0.01 钙信号通路 20 1.79 < 0.01 心肌细胞中的肾上腺素能信号 17 1.52 < 0.01 血管平滑肌收缩 15 1.34 < 0.01 致心律失常性右心室心肌病(ARVC) 11 0.98 < 0.01 年轻人的成年型糖尿病 7 0.63 < 0.01 唾液分泌 12 1.07 < 0.01 心肌收缩 11 0.98 < 0.01 昼夜节律 12 1.07 < 0.01 胆碱能突触 13 1.16 < 0.01 催产素信号通路 15 1.34 < 0.01 视黄醇代谢 9 0.80 < 0.01 胰腺分泌物 11 0.98 < 0.01 药物代谢-细胞色素P450 9 0.80 < 0.01 cGMP-PKG信号通路 15 1.34 < 0.05 蛋白质消化吸收 10 0.89 < 0.05 酪氨酸代谢 6 0.54 < 0.05 Ⅱ型糖尿病 7 0.63 < 0.05 苯丙胺成瘾 8 0.71 < 0.05 补体和凝血级联 8 0.71 < 0.05 癌症中的转录失调 14 1.25 < 0.05 胃酸分泌 8 0.71 < 0.05 肌动蛋白细胞骨架的调节 16 1.43 < 0.05 -
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