基于单细胞RNA测序的膀胱癌预后风险模型的构建与验证

目的基于单细胞RNA测序(scRNA-seq)生物信息学分析的预后相关的差异表达基因构建膀胱癌预后风险模型并验证。方法从基因表达综合(GEO)数据库中下载膀胱癌scRNA-seq数据集GSE135337、GSE129845, 数据更新时间分别为2022年、2019年;下载常规转录组数据集GSE13507(数据更新时间为2020年)中165例膀胱癌样本的表达谱及其生存信息。从癌症基因组图谱(TCGA)数据库中下载414例膀胱癌样本和19例癌旁样本的表达谱数据及405例膀胱癌患者的临床信息。采用R 4.1.2软件对GEO数据库中的10例膀胱癌单细胞样本进行质量控制及降维聚类并对其进行细胞注释;采用CellChat分析GEO数据库中单细胞数据的细胞间通信。采用单因素Cox比例风险模型分析筛选与膀胱癌预后相关的差异表达基因, 并使用LASSO-Cox回归分析构建预后风险模型, 计算风险评分。根据中位风险评分, 以TCGA数据集中膀胱癌患者为训练集, 将患者分为低风险组和高风险组;采用GEO数据库GSE13507数据集为验证集进行验证, 通过Kaplan-Meier分析比较训练集、验证集低风险组...     

基于脊髓层面的骨癌痛发生机制

骨癌痛动物模型的不断出现和日渐完善为癌痛机制研究提供了有效的工具,癌痛的病理机制异于炎性痛和神经病理性痛,具有其独特性。全文以骨癌动物模型为载体,综述骨癌痛发生的脊髓机制。     

INHBA在结直肠癌中的表达及其与微卫星状态和临床病理特征的关系

目的探讨INHBA在结直肠癌中的表达及其与微卫星状态、临床病理特征之间的关系。方法基于基因表达综合(GEO)数据库及基因表达谱交互分析(GEPIA)数据库进行生物信息学分析, 选定结直肠癌差异表达预后相关目标基因。收集2016年1月至2022年6月于锦州医科大学附属第三医院行手术治疗的107例结直肠癌患者蜡块组织, 制备组织芯片, 采用免疫组织化学法检测结直肠癌组织中微卫星状态[错配修复(MMR)蛋白MLH1、MSH2、MSH6、PMS2均为阳性表达为错配修复完整(pMMR), 代表微卫星低度不稳定或微卫星稳定;若其中任何一个指标为阴性, 则判定为错配修复缺陷(dMMR), 代表微卫星高度不稳定]和INHBA的表达情况。回顾性分析患者临床病理资料, 分析INHBA与微卫星状态及临床病理特征之间的关系。结果从GEO数据库筛选出结直肠癌3个数据集:GSE110223(13个癌组织、13个癌旁组织)、GSE110224(17个癌组织、17个癌旁组织)、GSE113513(14个癌组织、14个癌旁组织), 筛选出癌组织和癌旁组织间差异表达上调和下调的前50个基因, 经韦恩图分析3个数据集的交集...     

基于生物信息学分析的食管鳞状细胞癌关键枢纽基因的筛选及验证

[摘要] 目的: 采用多种生物信息学分析工具,筛选与食管鳞状细胞癌（esophageal squamous cell carcinoma, ESCC）发生发展相关的枢纽基因（Hub gene）,并分析其生物学功能。方法：选取GEO食管鳞状细胞癌芯片数据GSE100942 为研究对象,采用GEO2R软件对数据进行处理和分析,筛选差异表达基因,并通过生物信息学工具DAVID、String、Cytoscape 构建差异表达基因的蛋白互作网络并筛选Hub基因;应用GO及KEGG进行生物功能富集分析;同时通过网络工具MiRDB寻找可能调控Hub基因的miRNA,并构建Hub 基因-miRNA调控网络;利用GEPIA在线分析工具对筛选基因的表达和患者生存情况进行验证。结果：分析GSE100942 芯片数据共筛选出1229 个表达差异达2 倍以上及223 个表达差异达4 倍以上的差异基因,以及在食管癌组织中表达均上调的20 个Hub基因;功能富集分析显示这些差异基因主要富集到了癌症相关通路,并主要参与了细胞分裂及有丝核分裂等生物学过程;从Hub基因进一步鉴定了DLGAP5、BUB1B、TPX2、TTK、CDC20、CCNB2、AURKA、DEPDC1 为食管鳞状细胞癌相关的8 个关键Hub基因,他们参与了细胞增殖、细胞周期、信号通路等重要生物学过程。通过构建的miRNA调控网络分析,鉴定了CEP55、ECT2、NEK2、DEPDC1 及NUSAP1 等5 个Hub 基因受该网络高度调控。结论：基因芯片结合生物信息学方法能够有效分析与食管鳞状细胞癌发生发展相关的差异表达基因,筛选出的20 个Hub基因和其中的8 个关键基因可为进一步研究食管鳞状细胞癌发病的分子机制及分子标志物的筛选提供理论指导。     

利用基因表达谱筛选肝细胞癌中炎性微环境形成相关新基因的研究

目的:利用基因表达谱芯片筛选肝细胞癌(HCC)中的差异表达基因,并分析神经降压素(NTS)表达与HCC中炎性微环境形成的相互关系.方法:收集10例原发性HCC癌与癌旁组织,提取RNA进行Affymetrix全基因组表达谱芯片检测,同时整合GEO数据库中同芯片平台的亚洲人原发性HCC的mRNA表达谱数据30例,利用R2.9.0软件获得差异基因列表,应用MemCore(R)2.5,Pathway Studio(R)6.0和Ingenuity(R)1.0软件进行信号通路分析.最后,结合HE染色比较不同NTS表达水平的HCC标本中炎症反应强度.结果:所有RNA样品无降解,各芯片数据的信号强度分布均一.SMA聚类和信号通路分析结果显示40例HCC标本中存在一组独特亚群,呈现神经降压素(NTS)高表达,并伴有胞外间质重构、细胞粘附、白细胞移动、血管新生等生物学过程显著增强.HE染色证实NTS高表达标本中炎细胞浸润、纤维增生和血管生成明显高于NTS低表达的标本.结论:NTS高表达可能促进HCC中炎性环境的形成.     

RNF135在食管鳞状细胞癌中的表达及临床意义

目的 探讨环指蛋白135(RNF135)在食管鳞状细胞癌(ESCC)组织中的表达情况及临床意义。方法 收集2015年1月至2016年12月90例ESCC组织及癌旁组织。从高通量基因表达(GEO)数据库获取ESCC表达的数据集(GSE118037、GSE132867、GSE116958),采用在线工具GEO2R筛选ESCC组织与正常组织中具有差异表达的基因。对差异表达最显著的上调基因RNF135,采用Western blotting检测其在3组癌组织及癌旁组织中的表达,并采用免疫组化法检测RNF135在90例不同级别ESCC组织中的表达情况。采用Kaplan-Meier法分析不同RNF135表达患者的总生存时间(OS)。结果 3个数据集取交集后共筛选出ESCC中的差异表达基因93个,表达上调44个,表达下调49个。选择表达上调最显著的RNF135进行后续研究。Western blotting检测显示,与癌旁组织比较,RNF135在ESCC组织中的表达增加(P<0.01)。免疫组化染色显示,RNF135高表达率为56.7%(51/90),低表达率为43.3%(39/90)。RNF13...     

兔VX2腹膜转移癌模型的建立及转移特征研究

目的:建立大动物腹膜转移癌模型,鉴定其生物学行为.方法:36只新西兰大白兔分为开腹包埋、开腹穿刺及直接经皮穿刺种植三组(每组12只),每组又分为6只瘤块接种,6只悬液接种,接种VX2肿瘤.观察肿瘤生长状况,通过病理学和影像学检查分析局部区域及远处转移情况.结果:三种方法构建模型的成功率分别是100%(12/12)、91.7%(11/12)和58.3%(7/12),开腹法与经皮穿刺法相比有显著性差异(P＜0.05).接种2周后,形成典型溃疡型胃癌并腹膜转移癌表现,宿主衰竭,4周出现肺转移.病理学检查符合典型VX2组织学特点.结论:开腹包埋与开腹穿刺接种制作VX2兔腹膜转移癌模型简单,实验周期短,接种率高,其病理表现类似人类腹膜转移癌,为腹膜癌治疗的实验研究提供可靠的大型动物模型.     

基于高尔基体相关基因喉癌预后模型的构建

目的:基于TCGA数据库筛选预后相关的高尔基体相关基因,进一步构建喉癌预后的风险模型。方法:根据分子特征数据库获取高尔基体相关基因图谱。筛选TCGA数据库中喉癌和癌旁组织转录组数据中高尔基体相关基因表达谱,采用limma法对癌和癌旁数据进行差异分析。高尔基体相关基因表达谱结合临床数据进行单因素独立预后分析获取与预后相关的基因。差异基因与预后基因进行交叉分析,获得的基因再进行LASSO回归构建模型。Kaplan-Meier法分析高低风险组预后。ROC曲线验证该预测模型的可靠性。利用实时荧光定量PCR和免疫印迹实验鉴定喉癌和癌旁组织中基因表达的差异。结果:从MSigDB数据库获得1 659个高尔基体相关基因,结合TCGA数据库中喉癌数据,共获得1 084个高尔基体基因表达矩阵,通过差异分析共得到77个上调和190个下调高尔基体相关基因。通过对1 084个高尔基体相关基因进行单因素预后分析,得到了8个喉癌预后抑制因子和15个喉癌预后促进因子。通过交叉分析,最终得到1个在喉癌癌旁组织中高表达的预后抑制因子和5个喉癌组织中高表达的预后促进因子。对获得的6个基因进行LASSO回归分析构建模型,Kaplan-Meier法分析结果显示高风险组比低风险组预后差（P＜0.05）。ROC曲线验证该预测模型的可靠性较好。实时荧光定量PCR和免疫印迹实验验证TMC8在喉癌中表达低于癌旁组织,SLC35C1、PDGFA、GALNT2、ITGA5在喉癌中表达高于癌旁组织。结论:采用生物信息学分析方法结合实验验证,筛选并构建了一个喉癌预后的预测模型,为该疾病的研究和诊治提供新的思路和方法。     

肿瘤坏死因子受体超家族成员21在肝细胞癌中的表达及临床意义

目的 基于生物信息学分析探讨肿瘤坏死因子受体超家族成员21(TNFRSF21)在肝细胞癌(HCC)中的表达和预后价值、潜在的生物学功能及对免疫微环境的影响.方法 从高通量基因表达(GEO)数据库获取HCC细胞株数据集筛选共交集基因.从癌症基因组图谱(TCGA)数据库获取HCC样本数据信息,分析TNFRSF21表达与总生...     

长链非编码RNA EPB41L4A-AS2对乳腺癌的预后研究

目的 通过检测乳腺癌组织中EPB41L4A-AS2的表达,评价其临床病理特征与乳腺癌患者预后之间的关系。方法利用癌症基因组Meta分析、TCGA和GEO数据集,评价EPB41L4A-AS2的表达量与乳腺癌的临床组织特征之间的关系。利用Western blot实验,在乳腺癌细胞水平验证EPB41L4A-AS2与凋亡通路的关联性。结果 在Meta分析、TCGA和基因表达库(GEO)数据集的结果中发现,乳腺癌组织中EPB41L4A-AS2低表达,并且与不良的临床病理特征呈正相关。而在乳腺癌细胞系中的实验验证了EPB41L4A-AS2与细胞经典凋亡通路相关联。在GEO中进行Meta分析的预后研究中,发现EPB41L4A-AS2的高表达与良好的疾病预后有关。结论 EPB41L4A-AS2抑制了肿瘤的形成,对乳腺癌的临床预后分析有较高价值。     

Toppgene筛选肺腺癌候选疾病基因

背景与目的肺腺癌是危害人类健康最主要的肺癌类型之一,其发生机制仍不清楚。本研究利用生物信息学方法,筛选新的肺腺癌候选基因,为揭示肺腺癌发病机制提供依据。方法从GEO数据库中获得GSE10072和GSE7670两个数据集,然后利用dchip软件进行差异表达基因分析,将其获得的差异基因定义为检测基因集(testgeneset);采用genecard和Fable文献挖掘已知肺腺癌疾病基因,并将其定义为训练基因集(traingeneset);最后,利用Toppgene筛选肺腺癌候选基因,并通过荧光定量PCR对其获得的部分基因进行验证。结果获得一个含344个基因的检测基因集和含277个基因的训练基因集。采用Toppgene共获得36个候选疾病基因,其中21个基因则在肿瘤方面的研究几无报道。荧光定量PCR实验研究发现,CD36、PMAIP1及FABP4三个基因在A549细胞中均为下调表达,与芯片数据一致。结论Toppgene可发现新的肺腺癌候选疾病基因,为下一步发现特异性肺腺癌致病基因提供理论依据。     

利用GEO数据集分析USP51在胃癌中的表达及临床意义

目的:研究USP51在胃癌中的表达情况,探讨USP51表达与胃癌临床病理参数的相关性并预测USP51表达对胃癌预后评估的临床价值。方法:从人类肿瘤相关基因表达汇编(Gene Expression Omnibus,GEO)数据库下载胃癌样本数据集GSE62254的series matrix数据,只保留临床资料和生存信息完整的患者病例,共收录胃癌患者295例。分析不同USP51表达与胃癌临床病理参数的相关性,以及对胃癌预后的影响。采用基因集富集分析方法分析和预测受USP51调控的相关基因。结果:USP51表达水平与胃癌患者年龄、T 分期、pTNM分期及Lauren分型均有关（P均<0.05),与胃癌患者性别及N分期无关（P均>0.05）。随着USP51表达程度的增加,胃癌患者总生存时间(overall survival,OS)明显缩短（P＜0.001）。USP51高表达富集了黏着斑通路、钙离子信号通路、细胞黏附分子和细胞外基质等相关的基因通路（P＜0.05,FDR<0.25）。结论:在胃癌中,USP51高表达是一种预后不良因素,推测其可以作为判断胃癌患者预后的有效生物标志物以及治疗靶点。     

基于GEO数据库的胃肠上皮化生相关基因与通路的生物信息学分析

目的 挖掘胃黏膜肠化过程中的差异基因、探索其发病机制并验证差异基因是否在胃癌发生过程中持续发挥作用。方法 在美国国立生物技术信息中心（NCBI）的GEO数据库中检索正常胃黏膜肠化表达谱芯片,并通过GEO2R分析得到差异基因,以及在不同芯片数据中均差异表达的关键基因。将差异基因利用生物学信息注释数据库DAVID进行GO生物学过程富集分析和KEGG通路富集分析,探索正常胃组织向肠化转变的相关生物学通路。并通过TCGA数据库分析关键基因在胃癌组织中的差异变化,通过KMplotter分析关键基因与胃癌患者预后的关系。结果 检索到3个涉及正常胃黏膜组织发生肠化有关基因芯片,通过差异分析得到在肠化中差异表达的基因共1188个,其中ALDOB、CLCA1、CLDN7、DMBT1、KRT20、MTTP、OLFM4、REG3A和TFF3这9个关键基因在三个芯片中均差异表达。GO富集及KEGG通路分析显示,差异基因主要参与营养物质的消化吸收、蛋白质的水解与合成、物质转运调节等过程。TCGA数据库分析显示,上述9个关键基因在胃癌组织中亦具有差异变化,且通过KM plotter分析证实其与患者预后密切相关。结论 本研究获取了在肠化中异常表达的差异基因及其相关通路,并证实关键基因与胃癌患者预后密切相关。     

生物信息学方法筛选结肠癌相关长链非编码RNA

目的 筛选结肠癌中差异表达长链非编码RNA（lncRNA）并探讨其表达情况。方法 从NCBI（美国国立生物技术信息中心）公共数据平台Gene Expression Omnibus（GEO）下载结肠癌基因芯片数据GSE41328,包含10对结肠癌组织及正常组织,用微阵列显著性分析（SAM）软件输出差异表达基因,信息学网站对基因重注释得到lncRNA名称,然后用Gene Cluster和TreeView软件分析差异表达lncRNA数据,进一步验证其表达情况。结果 分析GSE41328发现,与正常组织相比,结肠癌共有66个lncRNA差异表达,其中22个高表达,44个低表达,结肠癌与正常组织表达值倍数均大于2或小于0.5,差异有统计学意义。结论 运用生物信息学方法筛选结肠癌相关lncRNA,可为寻找新的肿瘤标志物提供新的途径,但其结果需要进一步实验验证。     

Meta-analysis of Gene Expression Data Identifies Causal Genes for Prostate Cancer

Prostate cancer is a leading cause of death in male populations across the globe. With the advent of geneexpression arrays, many microarray studies have been conducted in prostate cancer, but the results have variedacross different studies. To better understand the genetic and biologic mechanisms of prostate cancer, we conducteda meta-analysis of two studies on prostate cancer. Eight key genes were identified to be differentially expressedwith progression. After gene co-expression analysis based on data from the GEO database, we obtained a coexpressedgene list which included 725 genes. Gene Ontology analysis revealed that these genes are involvedin actin filament-based processes, locomotion and cell morphogenesis. Further analysis of the gene list shouldprovide important clues for developing new prognostic markers and therapeutic targets.     

Amphiregulin anti-sense oligodeoxynucleotides inhibit growth and transformation of a human colon carcinoma cell line

Amphiregulin (AR) is a secreted heparin-binding growth factor that is structurally and functionally related to epidermal growth factor (EGF) and transforming growth factor a (TGFα). GEO cells are from a human colon cancer cell line that expresses high levels of AR protein and mRNA. To assess the role of AR in colon-cancer cell proliferation and transformation, 2 different anti-sense 20-mer phosphorothioate oligodeoxy-nucleotides (AR AS-1 and AR AS-2 S-oligos) complementary to the 5′ sequence of AR mRNA were synthesized. Both AR AS S-oligos were able to inhibit the anchorage-dependent growth (ADG) of GEO cells. The 2 AR AS S-oligos were equipotent when used in equimolar concentrations. In particular, a 40% growth inhibition was observed at a concentration of 10 μM, while a mis-sense S-oligo used as control had no effect on GEO cell growth. The AR AS-1 S-oligo used at the same concentration also inhibited by 40% the ³H-thymidine incorporation by DNA of GEO cells. The anchorage-independent growth (AIG) of GEO cells was even more significantly affected by AR AS S-oligo treatment. In fact, up to 80% inhibition of the AIG of GEO cells was observed when cells were treated with 10 μM of both AR AS S-oligos. Finally, the AR AS S-oligos were able to specifically inhibit AR protein expression in GEO cells, as assessed by immunocytochemistry. These data suggest that AR is involved in colon-cancer cell transformation, and that AR may represent a suitable target for gene therapy in human colon carcinomas. © 1995 Wiley-Liss, Inc.     

Protection against Whole Body -Irradiation Induced Oxidative Stress and Clastogenic Damage in Mice by Ginger Essential Oil

Radioprotective effects of ginger essential oil (GEO) on mortality, body weight alteration, hematological parameters, antioxidant status and chromosomal damage were studied in irradiated mice. Regression analysis of survival data in mice exposed to radiation yielded LD50/30 as 7.12 and 10.14 Gy for control (irradiation alone) and experimental (GEO-treated irradiated) mice, respectively, with a dose reduction factor (DRF) of 1.42. In mice exposed to whole-body gamma-irradiation (6 Gy), GEO pre-treatment at 100 and 500 mg/kg b.wt (orally) significantly ameliorated decreased hematological and immunological parameters. Radiation induced reduction in intestinal tissue antioxidant enzyme levels such as superoxide dismutase, catalase, glutathione peroxidase and glutathione was also reversed following administration of GEO. Tissue architecture of small intestine which was damaged following irradiation was improved upon administration of GEO. Anticlastogenic effects of GEO were studied by micronuclei assay, chromosomal aberration and alkaline gel electrophoresis assay. GEO significantly decreased the formation of micronuclei, increased the P/N ratio, inhibited the formation of chromosomal aberrations and protected agaisnt cellular DNA damage in bone marrow cells as revealed by comet assay. These results are supportive of use of GEO as a potential radioprotective compound.     

Antitumor activity of ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in human cancer cells with acquired resistance to antiepidermal growth factor receptor therapy.

PURPOSE: The epidermal growth factor receptor (EGFR) autocrine signaling pathway is involved in cancer development and progression. EGFR inhibitors such as C225 (cetuximab), a chimeric human-mouse anti-EGFR monoclonal antibody, and ZD1839 (gefitinib), a small molecule EGFR-selective tyrosine kinase inhibitor, are in advanced clinical development. The potential emergence of cancer cell resistance in EGFR-expressing cancers treated with EGFR inhibitors could determine lack of activity of these drugs in some cancer patients. Vascular endothelial growth factor (VEGF) is secreted by cancer cells and plays a key role in the regulation of tumor-induced endothelial cell proliferation and permeability. ZD6474 is a small molecule VEGF flk-1/KDR (VEGFR-2) tyrosine kinase inhibitor that also demonstrates inhibitory activity against EGFR tyrosine kinase. EXPERIMENTAL DESIGN: The antitumor activity of ZD1839, C225, and ZD6474 was tested in athymic mice bearing human GEO colon cancer xenografts. GEO cell lines resistant to EGFR inhibitors were established from GEO xenografts growing in mice treated chronically with ZD1839 or C225. Expression of EGFR was evaluated by flow cytometry. Expression of various proteins involved in intracellular cell signaling was assessed by Western blotting. Tumor growth data were evaluated for statistical significance using the Student's t test. All Ps were two-sided. RESULTS: Although chronic administration of optimal doses of C225 or ZD1839 efficiently blocked GEO tumor growth in the majority of mice, tumors slowly started to grow within 80-90 days, despite continuous treatment. In contrast, continuous treatment of mice bearing established GEO xenografts with ZD6474 resulted in efficient tumor growth inhibition for the entire duration of dosing (up to 150 days). ZD6474 activity was also determined in mice pretreated with ZD1839 or C225. When GEO growth was apparent after 4 weeks of treatment with EGFR inhibitors, mice were either re-treated with EGFR inhibitors or treated with ZD6474. GEO tumor growth was blocked only in mice treated with ZD6474, whereas tumor progression was observed in mice re-treated with C225 or ZD1839. GEO tumors growing during treatment with C225 or with ZD1839 were established as cell lines (GEO-C225-RES and GEO-ZD1839-RES, respectively). Cell membrane-associated EGFR expression was only slightly reduced in these cell lines compared with parental GEO cells. Western blotting revealed no major change in the expression of the EGFR ligand transforming growth factor alpha of bcl-2, bcl-xL, p53, p27, MDM-2, akt, activated phospho-akt, or mitogen-activated protein kinase. However, both GEO-C225-RES and GEO-ZD1839-RES cells exhibited a 5-10-fold increase in activated phospho-mitogen-activated protein kinase and in the expression of cyclooxygenase-2 and of VEGF compared with GEO cells. GEO-C225-RES and GEO-ZD1839-RES growth as xenografts in nude mice was not significantly affected by treatment with either C225 or ZD1839 but was efficiently inhibited by ZD6474. CONCLUSIONS: Long-term treatment of GEO xenografts with selective EGFR inhibitors results in the development of EGFR inhibitor-resistant cancer cells. Growth of EGFR inhibitor-resistant tumors can be inhibited by ZD6474. These data indicate that inhibition of VEGF signaling has potential as an anticancer strategy, even in tumors that are resistant to EGF inhibitors.     

32基因联合临床病理数据预测淋巴结阴性乳腺癌术后转移风险

目的探讨基因表达谱结合临床病理数据预测淋巴结阴性乳腺癌术后转移风险的方法。方法搜索GEO芯片数据库中乳腺癌根治术后、原发肿瘤≤5 cm、淋巴结阴性、未接受术前新辅助治疗的样本,将所有标本按照2∶1的比例随机分成训练组和验证组,选取训练组与无远处转移生存期（distant metastasis free survival,DMFS）密切相关的基因,以患者年龄、病理类型、肿瘤大小、内分泌治疗方式为变量拟合Cox回归模型,预测验证组患者远处转移风险,Kaplan-Meier法计算生存率和绘制生存曲线并行Log rank检验。结果本研究共纳入367例样本的6 214个基因的表达谱,训练组单基因生存分析表明32个基因探针的表达水平与淋巴结阴性乳腺癌术后DMFS密切相关（P<0.001）,根据32基因联合临床病理数据拟合的Cox比例风险模型计算验证组122例患者的预后指数,预后指数＞0.833为高风险组,中位DMFS为(4 026±382)天;预后指数≤0.833为低风险组,中位DMFS为(7 237±382)天,两组生存曲线的比较差异有统计学意义（χ²=5.900,P=0.015）。结论32基因表达联合临床病理数据进行淋巴结阴性乳腺癌术后转移风险的预测是可行且有效的。