基于GEO数据库的肝癌相关基因的筛选及验证

肝细胞癌(hepatocellular carcinoma,HCC)是中国高发的恶性肿瘤之一,识别肝细胞癌发生发展相关基因,对于深入研究肝癌发病机制和开发诊疗靶点均具有重要意义.本研究利用GEO2R工具从基因表达汇编数据库(Gene Expression Omnibus Database,GEO)筛选5个数据集中共有的差异表达基因作为潜在的肝癌相关基因.利用Metascape网站,对差异表达基因进行功能富集及信号通路分析.结合GEPIA(Gene Expres-sion Profiling Interaction Analysis)网站筛选具有临床意义的基因.利用荧光定量PCR技术验证与肝癌预后相关的差异表达基因,候选肝癌相关基因,为后续的深入研究奠定扎实的基础.结果显示,从5个数据集中共发现94个共有的差异表达基因.文献检索后发现24个基因与肝癌发生发展的关系少见文献报道,属于肝癌中未知功能基因.利用GEPIA分析癌症基因组图谱(the cancer genome atlas,TCGA)中数据后发现,GINS1在肝癌组织中高表达,与肝癌患者生存期呈负相关;CFHR4和DNASE1L3在肝癌组织中显著低表达,与肝癌患者生存期呈正相关.荧光定量PCR技术证实GINS1在81.3％的肝癌组织中呈现高表达,CFHR4和DNAS-E1L3分别在71.9％和93.8％肝癌组织中低表达.因此,本研究发现GINS1、CFHR4和DNASE 1L3在肝癌组织中显著差异表达,与肝癌患者的预后密切相关,可能作为潜在的判断肝癌患者预后的分子标志物和研发肝癌治疗的潜在靶标.     

应用抑制消减杂交分离雌、雄鸡胚性分化早期的差异表达基因

分离不同性别的鸡胚性分化早期差异表达基因,可为禽类性别决定和性分化机制研究提供基本信息。本研究分别以孵化3.5-6d的雌、雄鸡胚性腺为材料,利用抑制性消减杂交技术成功构建了雌-雄鸡胚间正、反向消减cDNA文库,并利用斑点印迹杂交从中筛选出了39个性别差异表达的阳性cDNA克隆。以持家基因GAPDH为参照指标检测消减文库的消减效率,结果发现两个文库的消减效率均高达25倍。插入片段PCR鉴定结果显示,消减文库中cDNA插入片段的长度主要分布于250-750bp之间。分别对雌、雄鸡胚消减文库中的252和168个cDNA克隆进行斑点杂交筛选,再随机从两个消减文库中共抽取39个阳性差异表达克隆进行序列测定及序列比对分析。结果表明:这39个cDNA克隆分别代表了定位于鸡不同染色体上的18个已知功能的基因和11个假定基因;参照哺乳动物同源基因的功能,所得的18个已知差异基因可能参与多种生物反应过程。用半定量RT-PCR方法对雌、雄鸡胚消减文库中各5个基因的表达情况进行进一步验证,发现除雌性库中的一个基因外其它9个基因均有较明显的性别差异表达。这些性别差异表达基因的获得为进一步研究鸡胚性腺发育中的基因表达调控奠定了基础     

P38和ERK1/2在肝细胞癌中的表达及意义

目的探讨p38（mitogen-activated protein kinase p38,p38）和细胞外信号调节激酶1/2（extracellular reg-ulated kinase 1/2,ERK1/2）在不同分化程度肝细胞癌（human hepatocellular carcinoma,HCC）中的表达情况,以及两者的相关性。方法通过免疫组化（Envision）法,检测52例肝癌组织及l0例癌旁正常肝脏组织中p38和ERK1/2的表达。结果：p38和ERK1/2在肝癌组织中均有表达,与正常组比较差异有显著性,且阳性率的高低与其分化程度有关,p38在肝癌组织中的表达随分化程度的增高阳性率降低（P〈0.05）;ERK1/2在中、低分化的肝癌组织分别与高分化相比差异具有显著性（P〈0.05）。但低分化与中分化的肝癌组织比较虽也有差异但无统计学意义（P〉0.05）。癌组织中p38和ERK1/2的表达呈中度正相关（r=0.703,P〈0.05）。结论HCC中,p38和ERK1/2表达和活性增加,且存在一定的相关性。     

肝细胞癌中HIF-1α与PHD2的表达及临床意义

目的 研究HIF-1α和PHD2在肝细胞癌（HCC）组织中的表达,并分析其与临床病理特征间的相关性及临床意义.方法 应用免疫组化Envision法检测103例HCC组织、14例癌旁组织及13例正常肝组织中HIF-1α和PHD2的表达.结果在HCC、癌旁组织、正常组织中HIF-1α阳性表达率分别为63.1%、35.7%、0%,差异显著（P≤0.05）,其阳性表达与肿瘤大小、脉管瘤栓、分化程度、TNM分期均有显著差异 （P〈0.05）.PHD2在HCC组织中表达阳性率明显低于正常肝组织 （P≤0.05）.HCC中PHD2表达水平下调与肿瘤大小、分化程度、TNM分期的差异有统计学意义（P〈0.05）.生存分析显示HIF-1α阳性组与阴性组2年生存率分别为53.8%（35/65）、81.6%（31/38）,差异有统计学意义（P〈0.05）.Cox多因素分析显示肿瘤大小、脉管瘤栓、HBsAg阳性及HIF-1α的表达是影响HCC的独立预后因素.HIF-1α和PHD2表达呈正相关（r=0.219,P〈0.05）.结论 HIF-1α和PHD2的异常表达与HCC的一些恶性进展临床病理特征相关,提示其可能参与肝癌的发生、发展、侵袭,有助于肿瘤的预后分析.     

人细胞差异表达基因cDNA消减杂交体系的建立

为克隆肺腺癌分化相关基因, 采用诱导分化与消减杂交相结合的策略, 建立了全反式维甲酸(RA)诱导前后人肺腺癌细胞系的cDNA消减文库, 得到124个cDNA消减克隆. 经加减法杂交差异筛选、DNA和RNA印迹、cDNA全序列测定和生物学功能分析, 分离到3个在人肺腺癌细胞系分化过程中由RA激活而特异表达的新的cDNA序列这一策略和技术路线适用于分离细胞中呈过量表达或表达抑制基因的cDNA克隆, 并具有反映细胞分化过程中基因表达动态变化特征和相对简便适用的特点.     

筛选差异表达基因和蛋白质的方法进展

分离和鉴定差异表达基因和蛋白质不仅有助于发现基因和蛋白质的功能,更有助于揭示某些疾病的发生机理.目前筛选差异表达基因的方法主要有差异显示PCR方法(differential display RT-PCR,DDRT-PCR)、消减杂交法(subtractive hybridization,SH)、基因芯片技术(DNA chip technique)和基因表达的系统分析(serial analysis of gene expression,SAGE)等,其中消减杂交法中又先后建立了代表性差异分析技术(representational difference analysis,RDA)、抑制消减杂交法(suppression subtractive hybridization,SSH)和获得全长基因的消减杂交法(full-length-gene-obtainable subtractive hybridization).筛选差异表达蛋白质的方法主要有双向电泳技术(two-dimentional gel electrophoresis)和噬菌体全套抗体库技术(phage display antibody repertoire library technique).这些方法各有特点,各有利弊,研究者可根据自己的需要选择适合于自己的方法.     

抑癌基因p53 在肝癌组织中的异常表达及临床意义

目的:探讨抑癌基因p53在肝癌组织中表达及临床意义,为肝癌的治疗提供新的思路。方法:回顾性分析了2006年1月～2009年8月收治的40例肝癌患者的临床资料,采用免疫组织化学法测定肝癌组织、癌旁组织和正常组织中抑癌基因p53的表达,并分析不同肿瘤分化程度患者p53的表达。结果:p53阳性表达率在在肝癌组、癌旁组分别为47.5%和2.5%,在正常组未检测到p53阳性表达,三组之间比较有显著性差异(x2=6.515,P=0.024);高分化癌P53蛋白阳性表达率低,中低分化癌P53蛋白阳性表达率高,组比较均有显著性差异(P<0.05),中、低分化组之间比较差异无统计学意义(P>0.05)。结论:在肝癌组织中存在p53基因的高表达,其阳性表达与肿瘤分化程度有关。     

应用抑制性消减杂交分析全反式维甲酸和1,25-二羟维生素D_3诱导结肠癌细胞的相关基因

 为探讨结肠癌细胞诱导分化的机制 ,采用抑制性消减杂交 (suppression subtractivehybridization,SSH)研究联合使用全反式维甲酸和 1 ,2 5-二羟维生素 D3诱导分化结肠癌 Lo Vo细胞前、后差异表达的基因 .经比较消减 c DNA文库的序列与基因库的序列 ,发现 :有 1个基因的序列与正常鳞状上皮细胞中的 1个表达序列标签 (expressed sequence tag,EST)高度同源 ,同时发现6个新 EST (基因库登录号为 AW2 66492、AW2 66493、AW2 66494、AW58751 8、AW58751 9和AW58752 0 ) .说明诱导分化涉及到多个基因的表达 ,结肠癌的发生是多基因综合作用的结果 .进一步研究这些基因和 EST的功能对于结肠癌的防治将有重要意义 .     

RNA干扰肝细胞肝癌中PECAM-1的表达对肿瘤细胞侵袭及增殖的影响

目的:探讨PECAM-1在肝细胞肝癌(Hepatocellular carcinoma,HCC)组织中的表达及意义。方法:选择2013年5月-2015年6月在我院接受治疗的HCC患者100例,收集肝癌患者HCC组织及癌旁组织,另选取100例正常肝脏组织作为对照组。应用免疫组织化学法检测PECAM-1在肝癌组织、癌旁组织以及正常肝脏组织中的阳性表达。利用小分子干扰RNA技术(si RNA)构建低表达的PECAM-1,并转染至肝癌细胞中抑制PECAM-1的表达。应用Transwell小室法检测肝癌细胞的侵袭能力,CCK-8法检测肝癌细胞的增殖能力。结果:PECAM-1在肝癌组织、癌旁组织及正常肝脏组织中呈不同程度阳性表达(P0.05);PECAM-1在肝癌组织及癌旁组织中的表达显著高于正常肝脏组织,差异具有统计学意义(P0.05);PECAM-1在肝癌组织中的表达显著高于癌旁组织,差异具有统计学意义(P0.05);转染si RNA PECAM-1后,肝癌细胞中PECAM-1 m RNA的表达水平明显下降,PECAM-1蛋白表达也明显降低,差异具有统计学意义(P0.05);转染si RNA PECAM-1后,肝癌细胞侵袭及增殖能力明显降低,差异具有统计学意义(P0.001)。结论:PECAM-1在肝癌患者血清中高表达,PECAM-1 si RNA能够抑制肝癌细胞的侵袭及增殖能力,提示PECAM-1可作为预测肝癌发生及发展的临床指标。     

Bmi1 在肝癌组织中的表达及其与细胞增殖和凋亡的关系

目的:探讨Bmi1在肝细胞肝癌(HCC)中的表达及与增殖和凋亡的关系。方法:收集HCC标本54例及相应的癌旁组织,10例正常肝组织标本,采用免疫组织化学EnVision二步法显示Bmi1的表达并结合增殖与凋亡特征进行分析。结果:在肝癌组织、癌旁组织细胞中Bmi1表达定位于胞核中,阳性表达率分别为79.6%(43/54),31.2%(17/54),10例正常肝组织未见表达,3组差异有统计学意义(P<0.005)。HCC中Bmi1的高表达与年龄、性别、肿瘤大小、肿瘤数目、临床TNM分期、是否有肝硬化及是否有HBsAg感染无显著相关(P>0.05),但与组织学分级有关,高、中分化组Bmi1表达率显著高于低分化组(P<0.05)。肝癌Bmi1阳性组增殖指数(PI)(50.3±21.4)%显著高于阴性表达组(17.3±7.1)%(P<0.05),凋亡指数(AI)无明显差异(P>0.05)。结论:Bmi1在HCC中高表达,其表达增高可能与HCC的进展相关。     

生物信息学分析筛选结直肠癌靶基因及评估预后价值

为寻找与结直肠癌发展和预后相关的潜在关键基因及信号通路。从美国国立信息中心NCBI的GEO数据库获得结直肠癌基因表达数据集GSE106582,通过PCA对样本进行分组,利用GEO2R进行综合分析,筛选结直肠癌与癌旁对照组的差异表达基因;通过DAVID在线工具对差异表达基因进行GO本体分析和KEGG通路富集分析,初步分析差异表达基因的生物学作用;基于STRING数据库对差异表达基因进行蛋白质相互作用网络分析,利用Cytoscape软件进行可视化并筛选关键基因;用生存分析和ROC曲线诊断对关键基因进行鉴定并通过数据集GSE21510进行验证。共鉴定出199个差异表达基因,其中53个为上调基因,146个为下调基因;上调的差异表达基因主要富集在与胶原蛋白分解代谢过程、细胞外基质分解、细胞外基质受体相互作用和PI3K/AKT信号通路等生物学过程;下调的差异表达基因主要富集在碳酸氢盐运输、一碳代谢过程、矿物质吸收、药物代谢-细胞色素P450和氮代谢通路等生物学过程;MCODE分析、生存分析和ROC诊断共发现3个基因分别为BGN、COL1A2和TIMP1可能与结直肠癌的发生发展有关,它们在肿瘤组织中的异常高表达与患者较差的生存期呈正相关,GSE21510的验证结果与GSE106582的分析结果相同。本研究采用生物信息学方法对CRC基因芯片数据进行挖掘,从基因水平探讨CRC潜在的发病机制、肿瘤标志物的及患者预后分子的筛选,以及可能的药物治疗靶点提供了一定的参考价值和理论基础。     

Affymetrix基因表达谱芯片在新疆维吾尔族与汉族胰腺癌组织样本间差异表达基因筛选中的运用

目的:运用基因表达谱芯片筛选并分析新疆维吾尔族与汉族胰腺癌组织样本间的差异表达基因。方法:收集我院2014年1月至2016年6月间行手术切除的维吾尔族与汉族胰腺导管细胞癌组织并提取总RNA,选取经Nanodrop 2000与Agilent 2100仪器质检合格的样本总RNA采用Affymetrix基因表达谱芯片筛选出差异表达基因并绘制统计图,运用基因本体(GO)分析及信号通路(Pathway)分析对这些差异表达基因的生物信息进行汇总分析。结果:通过基因表达谱芯片分析,新疆维吾尔族与汉族胰腺癌组织样本间共检测到1063个基因存在差异表达,在维吾尔族胰腺癌标本中显著上调表达的基因共281个,差异表达倍数最高的为IGLV1-44基因(差异倍数:9.99)下调表达的基因共782个,差异表达倍数最高的为CPB1基因(差异倍数:33.76);在Gene Ontology数据库中共检索到815个上述差异表达基因具有明确的GO分类,差异表达倍数最高的为CPB1基因(差异倍数:33.76);Pathway分析中共检测到30条信号通路包含有上述差异表达基因,共涉及196个基因,其中以FAK信号通路差异表达基因富集程度最高,差异表达倍数最高的基因为COL11A1基因(差异倍数:5.02)。结论:基因表达谱芯片分析结果显示,在新疆维吾尔族与汉族胰腺癌组织样本间存在大量的差异表达基因,这些基因与胰腺癌的增殖分化、侵袭转移及多药耐药等特性密切相关,且参与了多条生物体内重要信号转导通路的调控。     

Gene expression signature of human HepG2 cell line

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is associated with various clinico-pathological characteristics such as genetic mutations and viral infections. Therefore, numerous laboratories look out for identifying always new putative markers for the improvement of HCC diagnosis/prognosis. Many molecular profiling studies investigated gene expression changes related to HCC. HepG2 represents a pure cell line of human liver carcinoma, often used as HCC model due to the absence of viral infection. In this study we compare gene expression profiles associated with HepG2 (as HCC model) and normal hepatocyte cells by microarray technology. Hierarchical cluster analysis of genes evidenced that 2646 genes significantly down-regulated in HepG2 cells compared to hepatocytes whereas a further 3586 genes significantly up-regulated. By using the Ingenuity Pathway Analysis (IPA) program, we have classified the genes that were differently expressed and studied the functional networks correlating these genes in the complete human interactome. Moreover, to confirm the differentially expressed genes as well as the reliability of our microarray data, we performed a quantitative Real time RT-PCR analysis on 9 up-regulated and 11 down-regulated genes, respectively. In conclusion this work i) provides a gene signature of human hepatoma cells showing genes that change their expression as a consequence of liver cancer in the absence of any genetic mutations or viral infection, ii) evidences new differently expressed genes found in our signature compared to previous published studies and iii) suggests some genes on which to focus future studies to understand if they can be used to improve the HCC prognosis/diagnosis.     

小鼠胚胎干细胞分化为肝细胞的基因芯片分析

弄清胚胎肝脏发育的分化调节机制,对指导干细胞在肝再生中的应用以及研究肝分化相关疾病分子机制具有重要意义．胚胎干细胞的全能性使得体外建立肝向分化模型成为可能,采用单层贴壁培养方式,分阶段加入成纤维细胞生长因子(FGF)、肝细胞生长因子(HGF)、制瘤素(OSM)等因子,诱导小鼠胚胎干细胞D3(mESC-D3)的肝向分化．分化细胞在光镜和电镜下呈现肝样细胞形态,RT-PCR、细胞免疫荧光检测以及PAS染色分析表明,这些细胞具有肝细胞特征性的基因表达和生化功能．采用干细胞分化相关基因芯片比较早期肝定向分化前后的基因表达差异,结果显示,48个差异表达基因中(大于2倍),20个上调、28个下调．进一步的生物信息学分析发现,它们集中体现在细胞外基质、细胞连接、FGF、BMP分子及Notch、Wnt信号通路上,提示这些改变可能与胚胎早期的肝向分化密切相关．     

Toxicogenomics of A375 human malignant melanoma cells treated with arbutin

Although arbutin is a natural product and widely used as an ingredient in skin care products, its effect on the gene expression level of human skin with malignant melanoma cells is rarely reported. We aim to investigate the genotoxic effect of arbutin on the differential gene expression profiling in A375 human malignant melanoma cells through its effect on tumorigenesis and related side-effect. The DNA microarray analysis provided the differential gene expression pattern of arbutin-treated A375 cells with the significant changes of 324 differentially expressed genes, containing 88 up-regulated genes and 236 down-regulated genes. The gene ontology of differentially expressed genes was classified as belonging to cellular component, molecular function and biological process. In addition, four down-regulated genes of AKT1, CLECSF7, FGFR3, and LRP6 served as candidate genes and correlated to suppress the biological processes in the cell cycle of cancer progression and in the downstream signaling pathways of malignancy of melanocytic tumorigenesis.     

Nano-LC-MS/MS based proteomics of hepatocellular carcinoma cells compared to Chang liver cells and tanshinone IIA induction

Human hepatocellular carcinoma (HCC) is one of the most malignant tumors, being particularly induced by unregulated growth and metastasis, and is a leading cause of death and major health problems in many countries. We report here the identification of 167 differentially expressed proteins between HCC (MHCC97-H) cells and Chang liver cells using enhanced nano-liquid chromatography/mass spectrometry (LC/MS). The most relevant pathways of differentially expressed proteins are involved in cytoskeleton organization, stress defense, and energy homeostasis etc. Moreover, of the identified proteins, there are 59 known or putative membrane-associated proteins with multitransmembrane domains confirmed by bioinformatic analysis. These proteins may be associated with cancer, reflecting tumorigenesis of HCC, and would be useful for the development of diagnostic and subsequently pharmaceutical targets of HCC. In addition, we identify a total of 41 proteins that are found to be up- or down-regulated following tanshinone IIA treatment for MHCC97H cells in a time-depended manner. Also, several proteins that are involved in actin cytoskeleton and stress resistance are mainly down-regulated, whereas proteins associated with cell redox homeostasis, mitochondrial, and microtubule-based movement are identified as mostly up-regulated after the treatment. Determination of functional roles of those differentially expressed proteins will enable further understanding of the mechanism of HCC tumorigenesis and exploration of new drugs for therapeutic intervention.     

Investigation of key microRNAs associated with hepatocellular carcinoma using small RNA-seq data

To identify key microRNAs (miRNAs) associated with hepatocellular carcinoma (HCC) using small RNA-seq data. Small RNA-seq data for two HCC samples and two normal samples were downloaded from NCBI Gene Expression Omnibus. MiRNAs were identified through database search. Differentially expressed miRNAs were screened out with t test and their target genes were retrieved. Functional enrichment analysis was performed to uncover their biological functions. Regulatory networks and core metabolic networks were also constructed to present the global patterns. In addition, new miRNAs and their target genes were predicted. A total of 59 differentially expressed miRNAs were obtained, 12 up-regulated and 47 down-regulated. A total of 3,306 target genes were retrieved for eight miRNAs. Pathway enrichment analysis for the target genes showed that “pathways in cancer” and “MAPK signaling pathway” were significantly over-represented. Functional enrichment analysis found that “biological regulation” and “macromolecule modification” were significantly related to the target genes. Two regulatory networks were constructed for up- and down-regulated differentially expressed miRNAs with information from Ingenuity Pathway Analysis database. Two metabolic networks were also established based upon “pathways in cancer” and “MAPK signaling pathway”, consisting of miRNAs, target genes, compounds and others genes. Moreover, a number of new miRNAs and relevant target genes were predicted. Our study discloses a number of miRNAs as well as genes which may be involved in the development of HCC and these findings are beneficial in guiding future researches.     

Establishment of mouse teratocarcinomas stem cells line and screening genes responsible for malignancy

The sequential transplantation of embryonal carcinoma cells in vivo can accelerate the growth and malignancy of teratocarcinomas. However, the possible molecular mechanisms in this process that reflect cancer formation in the early stage are largely unknown and. To identify which genes are associated with the changes of malignancy of teratocarcinomas, we established a tumorigenesis model in which teratocarcinoma were induced via injecting embryonic stem cells into immuno-deficiency mice, isolating teratocarcinoma stem cell from a teratocarcinoma in serum-free culture medium and injecting teratocarcinoma stem cells into immune-deficient mice continuously. By using high-throughput deep sequence technology, we identified 26 differentially expressed genes related to the changes of characteristics of teratocarcinoma stem cell in which 18 out of 26 genes were down-regulated and 8 genes were up-regulated. Among these genes, several tumor-related genes such as Gata3, Arnt and Tdgf1, epigenetic associated genes such as PHC1 and Uty were identified. Pathway enrichment analysis result revealed that Wnt signaling pathway, primary immunodeficiency pathway, antigen processing and presentation pathway and allograft rejection pathway were involved in the teratocarcinoma tumorigenesis (corrected p value<0.05). In summary, our study established a tumorigenesis model and proposed some candidate genes and signaling pathways that may play a key role in the early stage of cancer occurrence.     

Gene Expression Profiling Unravels Cancer-Related Hepatic Molecular Signatures in Steatohepatitis but Not in Steatosis

Background

Pathogenesis and factors for determining progression of alcoholic and non-alcoholic steatosis to steatohepatitis with risk of further progression to liver cirrhosis and cancer are poorly understood. In the present study, we aimed to identify potential molecular signatures for discrimination of steatohepatitis from steatosis.

Methodology and Results

Global microarray gene expression analysis was applied to unravel differentially expressed genes between steatohepatitis compared to steatosis and control samples. For functional annotation as well as the identification of disease-relevant biological processes of the differentially expressed genes the gene ontology (GO) database was used. Selected candidate genes (n = 46) were validated in 87 human liver samples from two sample cohorts by quantitative real-time PCR (qRT-PCR). The GO analysis revealed that genes down-regulated in steatohepatitis were mainly involved in metabolic processes. Genes up-regulated in steatohepatitis samples were associated with cancer progression and proliferation. In surgical liver resection samples, 39 genes and in percutaneous liver biopsies, 30 genes were significantly up-regulated in steatohepatitis. Furthermore, immunohistochemical investigation of human liver tissue revealed a significant increase of AKR1B10 protein expression in steatohepatitis.

Conclusions

The development of steatohepatitis is characterized by distinct molecular changes. The most striking examples in this respect were KRT23 and AKR1B10, which we found to be highly differentially expressed in steatohepatitis compared to steatosis and normal liver. We propose that KRT23 and AKR1B10 may serve as future potential biomarkers for steatohepatitis as well as markers for progression to HCC.