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结直肠癌是一种常见的恶性肿瘤,其致死率在恶性肿瘤中占第3位,仅次于肺癌和乳腺癌[1]。目前,放疗在结直肠癌的治疗过程中发挥着不可替代的作用[2]。然而,结直肠癌对放疗的抗性严重地影响了放疗患者的治疗效果与生存质量,其放疗抗性机制尚不明确[3-4]。基因表达数据库(Gene Expression Omnibus,GEO)是当今较大、且全面的公共基因表达数据库,为基因研究提供了大量的高通量数据[5]。本研究中我们主要利用GEO数据库筛选结直肠癌辐射耐受细胞中的差异表达基因,从分子水平上初步探讨与结直肠癌抗辐射相关的潜在基因,为下一步的实验研究提供理论基础。
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经过数据归一化处理,每组样本数据的中位值保持一致(归一化后中位值为52.48),消除了因个体基因组间不同所造成的基因间的差别,显示出良好的标准化程度,使得后续数据分析能够获得真实的处理结果。
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应用R语言中的limma包共筛选出差异基因101个,其中67个基因上调,占66.34%;34个基因下调,占33.66%。对前15个差异基因进行聚类分析结果见图 1所示,上调基因和下调基因中所对应的前15个差异基因可以被明显识别并加以区分。
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GO富集分析结果显示,差异基因富集在与细胞迁移、DNA复制、血管收缩等相关的生物学过程中,结果见图 2。KEGG通路分析显示,经过功能注释的差异基因主要富集在乏氧诱导因子1信号通路(VEGFA、EGLN3、HK2、ENO2、PDK1)、胆固醇代谢(SOAT1、LRP1、LDLR)等相关通路上。
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结果显示,PPI网络主要由122个基因和123种相互作用构成。同时,为了描述网络中存在的重要节点,我们采用点度中心性[16]、中介中心性[17]等方法,筛选出排名前10的基因,结果见表 1。通过对得到的Top基因取交集,最终得到在PPI网络中发挥关键作用的6个关键基因为NDRG1、PAG1、LRP1、PIM1、LDLR和PLAUR基因。
基因名 点度中心性 基因名 点度中心性 NDRG1 18 NDRG1 29.78 PAG1 16 LDLR 29 ENO2 12 HSPA5 25.95 RGS2 10 LRP1 24.87 LRP1 9 PIM1 25.03 PIM1 8 HSP90AA1 23.4 VEGFA 8 PAG1 22.57 LDLR 7 PLAUR 22.45 PLAUR 7 EFNA1 22.33 EPHA2 6 EPHA3 22.28 表 1 蛋白相互作用网络中根据节点中心度筛选的前10个基因
Table 1. The top 10 genes in protein-protein interaction network Network topology parameters
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结果显示,在人结肠癌HCT16细胞中,NDRG1、PAG1、LRP1、PIM1、LDLR和PLAUR等潜在抗性基因接受照射后,mRNA表达量显著上升,与照射前相比,差异均有统计学意义(t=49.981,P < 0.01;t=26.420、28.698、21.358、23.545,均P < 0.05;t=50.601,P < 0.01)。虽然这些抗性基因在照射后的mRNA相对表达水平与生物信息学预测结果相一致,但仍略有差别。实时荧光定量PCR实验证实,NDRG1和PLAUR(t=49.981、50.601,均P < 0.01)的mRNA相对表达水平在照射后变化明显,约为3倍差异的表达量变化,而基因芯片结果显示的差异变化明显的抗性基因为NDRG1和PAG1,差异倍数分别为2.60倍和1.96倍。结果见图 3。
应用生物信息学确定结直肠癌辐射抗性细胞的差异表达基因
Identification of genes for radiation resistance in colorectal cancer cells using bioinformatics analysis
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摘要:
目的基于生物信息学的方法,筛选结直肠癌辐射抗性细胞中的差异表达基因,从分子水平上初步探讨与结直肠癌抗辐射相关的潜在基因。 方法从基因芯片公共数据库(GEO)中下载耐辐射的结直肠癌细胞基因表达谱数据(GSE43206),并利用R语言中的limma包进行差异基因筛选。对差异基因中的编码基因分别进行基因本体论(GO)富集分析、京都基因和基因组百科全书(KEGG)通路分析以及蛋白相互作用(PPI)分析,进一步筛选出PPI网络中的关键基因。通过实时荧光定量PCR实验确定5 Gy γ射线照射后人结肠癌HCT116细胞中关键基因的mRNA相对表达水平。采用Student t-test检验进行统计学分析,P < 0.05表示差异有统计学意义。 结果共筛选出101个差异基因,包含67个上调基因,34个下调基因。GO富集分析发现这些差异基因在细胞迁移、DNA复制等生物学过程中富集。KEGG通路分析证实这些差异基因主要富集在乏氧诱导因子1信号通路。通过构建PPI网络,筛选出NDRG1、PAG1、LRP1、PIM1、LDLR和PLAUR共6个与结直肠癌抗辐射相关的潜在基因。实时荧光定量PCR实验结果显示,与照射前比较,照射后人结肠癌HCT116细胞中NDRG1、PAG1、LRP1、PIM1、LDLR和NDRG1、PAG1、LRP1、PIM1、LDLR关键基因的mRNA表达量显著上升,差异均有统计学意义(t=49.981,P < 0.01;t=26.420、28.698、21.358、23.545,均P < 0.05;t=50.601,P < 0.01)。 结论利用生物信息学能够快速地筛选出与结直肠癌抗辐射相关的潜在基因,且潜在基因在结直肠癌HCT116细胞中差异表达。 Abstract:ObjectiveTo preliminarily explore potential genes related to radiation resistance in colorectal cancer at the molecular level, we employed bioinformatics to screen different expression genes for radiation resistance in colorectal cancer cells. MethodsThe comparison between the gene expression levels of radiation resistance colorectal cancer cell lines and parental cell lines was downloaded from the Gene Expression Omnibus(GEO) database. The differentially expressed genes(DEGs) were screened by using the R Programming Language and were analyzed through Gene Ontology(GO) functional enrichment analysis and kyoto encyclopedia of genes and genomes(KEGG) pathway analysis and by using protein-protein interaction(PPI) networks. The hub genes were obtained on the basis of a PPI network. The mRNA relative expression level of the hub genes was verified via quantitative real-time polymerase chain reaction in HCT116 after radiation. The statistical significance of the results was analyzed via student t-test. ResultsA total of 101 DEGs were found in GSE43206, including 67 upregulated genes and 34 downregulated genes. The GO enrichment analysis suggested that these DEGs are enriched in biological processes, including cell migration and DNA replication. KEGG pathway analysis indicated that these DEGs were mainly enriched in the hypoxia inducible factor-1 signaling pathway. Six radiation resistance genes with high connectivity were identified on the basis of the PPI networks, including NDRG1, PAG1, LRP1, PIM1, LDLR, and PLAUR. Quantitative real-time polymerase chain reation verified that the expression levels of hub genes were markedly up-regulated in HCT116 after radiation, including NDRG1、PAG1、LRP1、PIM1、LDLR and PLAUR (t=49.981, P < 0.01; t=26.420, 28.698, 21.358, 23.545, all P < 0.05; t=50.601, P < 0.01). ConclusionsThe use of bioinformatics enabled effectively screening radiation resistance genes in colorectal cancer, which can be used for further researches. The molecular biology experiments confirmed the differential expression of potential genes after irradiation in colorectal cancer cell HCT116. -
Key words:
- Colorectal neoplasms /
- Radiation resistance /
- Different expression genes /
- Bioinformatics
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表 1 蛋白相互作用网络中根据节点中心度筛选的前10个基因
Table 1. The top 10 genes in protein-protein interaction network Network topology parameters
基因名 点度中心性 基因名 点度中心性 NDRG1 18 NDRG1 29.78 PAG1 16 LDLR 29 ENO2 12 HSPA5 25.95 RGS2 10 LRP1 24.87 LRP1 9 PIM1 25.03 PIM1 8 HSP90AA1 23.4 VEGFA 8 PAG1 22.57 LDLR 7 PLAUR 22.45 PLAUR 7 EFNA1 22.33 EPHA2 6 EPHA3 22.28 -
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