Effects of Euphorbiasteroid on gene expression in lung cancer cells based on gene chip

Objective Using gene chip technology to explore the molecular mechanism of euphorbiasteroid in the treatment of non-small cell lung cancer(NSCLC).Methods A549 cells were used as the in vitro model,and they were randomly divided into control group and different concentrations of euphorbiasteroid administration groups.Each group had 3 duplicate wells,after the cells were cultured in vitro,the cell viability was evaluated by CCK-8 method.Gene chip technology was used to screen the differentially expressed genes(DEGs)between the control group and the euphorbiasteroid administration group.The differential genes were further analyzed for Gene Ontology(GO)function enrichment and Kyoto Encyclopedia of Genes and Genome(KEGG)pathway enrichment analysis.The STRING online analysis platform combined with Cytoscape software to construct a target protein interaction(PPI)network and perform topological analysis to screen key targets,and use Real-time PCR(RT-PCR)and molecular docking technology to verify key targets.Results According to the analysis of gene chip data,276 differentially expressed genes were screened,including 117 up-regulated genes and 159 down-regulated genes.GO analysis showed that differentially expressed genes were mainly involved in cell division,cell proliferation,cell cycle and other processes,involving protein binding,protein kinase binding and other functions,and were mainly distributed in nucleoplasm,chromosomes and other parts.KEGG signaling pathway analysis showed that differentially expressed genes were involved in cell cycle,pyrimidine metabolism,p53 signaling pathway and other pathways.PPI network analysis showed that CCNA2,TOP2A,CCNB1,CDC20,and RRM2 may be the key targets of euphorbiasteroid in the treatment of NSCLC.RT-PCR results showed that the expressions of CCNA2,TOP2A,CCNB1,CDC20,and RRM2 were significantly down-regulated in the euphorbiasteroid administered group,which was consistent with the gene chip results.Molecular docking results showed that euphorbiasteroid had good affinity with key targets and could bind spontaneously and stably.Conclusion The combination of gene chip,RT-PCR technology and molecular docking technology can find out the differential genes after the intervention of euphorbiasteroid,which can be used to explore the mechanism of euphorbiasteroid in the treatment of NSCLC.

Microarray-Based Differential Expression Monitoring of 79 Novel Genes in Human Fetal Tissues

ESTs fragments which represents corresponding novel genes were obtained by sequencing and bioinformatics analysis of human fet al kidney cDNA library. Microarray was prepared by using these novel EST fragmen ts by automatic spotting. Expression patters of 79 ESTs of novel genes from huma n fetal kidney were analyzed in fetal brain and fetal heart tissues of 20\|week\ | and 26\|week\|age fetus by performing of cDNA chip hybridization. This provide s clues for studying exact functions of the novel genes. 8 genes were obtained w hich were expressed differentially in the fetal brain and heart of 20\|week\| an d 26\|week\|age respectively. Then differentially expressed genes were identifie d by Northern analysis. The more exact function of the novel genes is under stud y.

Gene expression profiles associated with osteoblasts differentiated from bone marrow stromal cells

Objective:To study the changes of gene expression profiles associated with osteoblasts differentiated from rat bone marrow stromal cells in vitro by gene chip technique.Methods:rat Rone marrow stromal cells were isolated and cultured,and differentiation was induced by dexamethasone,β-glycerol phosphate and vitamin C.Cellular mRNA was extracted and reverse transcribed into cDNA,thus related genes expression differences were detected by gene expression profile chip.Results:Calcifying nodules were visible in the induced cells.There were27.7%genes expressed differentially,three times more than the normal and induced cells,and some genes were related to transcription,translation,glycosylation modification.Extracellular matrix,signal molecules and metabolism were up—regulated.Conclusions:The gene chip technique can be used to detect the multi-gene different expression in the differentiationinduceed rat BMSCs,and these differentially expressed genes are necessary genes related to rat BMSCs proliferation and induction of osteoblastic differentiation.

ANALYSES ON DIFFERENTIALLY EXPRESSED GENES ASSOCIATED WITH HUMAN BREAST CANCER

Objective： To investigate the molecular etiology of breast cancer by way of studying the differential expression and initial function of the related genes in the occurrence and development of breast cancer. Methods： Two hundred and eighty-eight human tumor related genes were chosen for preparation of the oligochips probe, mRNA was extracted from 16 breast cancer tissues and the corresponding normal breast tissues, and cDNA probe was prepared through reverse-transcription and hybridized with the gene chip. A laser focused fluorescent scanner was used to scan the chip.The different gene expressions were thereafter automatically compared and analyzed between the two sample groups. Cy3/Cy5〉3.5 meant significant up-regulation. Cy3/Cy5〈0.25 meant significant down-regulation. Results： The comparison between the breast cancer tissues and their corresponding normal tissues showed that 84 genes had differential expression in the Chip. Among the differently expressed genes, there were 4 genes with significant down-regulation and 6 with significant up-regulation. Compared with normal breast tissues, differentially expressed genes did partially exist in the breast cancer tissues. Conclusion： Changes in multi-gene expression regulations take place during the occurrence and development of breast cancer; and the research on related genes can help understanding the mechanism of tumor occurrence.

Active Methyl Cycle and Transfer Related Gene Expression in Response to Drought Stress in Rice Leaves

Three rice varieties, Zhonghan 3, Shanyou 63 and Aizizhan, were used as materials in detecting differential active methyl cycle and transfer related gene expression in response to drought stress. The experiment was performed by gene chip and mRNA differential display technologies under the conditions of drought simulated with 10% PEG6000 solution. The results indicated that the methyl cycle could be activated in the leaves of Zhonghan 3 and Shanyou 63 but inhibited in the leaves of Aizizhan under drought stress. Furthermore, drought stress could induce the expression of a large number of methyltransferase genes, especially the transcription of Rubisco protein methylation related genes, which are beneficial for prevention of Rubisco protein oxidation and degradation, and drought stress could inhibit the transcription of DNA methyltransferase genes and histone methyltransferase genes. This result confirmed that the active methyl cycle and transfer related genes were involved in rice drought resistance.

Study on Tibetan Chicken embryonic adaptability to chronic hypoxia by revealing differential gene expression in heart tissue

Oxygen concentration is essential for appropriate metabolism.Hypoxia can exert a significant impact on physiological alteration of the cell and organism.Tibetan Chicken(Gallus gallus) is a Chinese indigenous breed inhabiting in Tibetan areas,which is also a chicken breed living at high altitude for the longest time in the world.It has developed an adaptive mechanism to hypoxia,which is demonstrated by that Tibetan Chicken has much higher hatchability than low-land chicken breeds in high-altitude areas of Tibet.In the present study,Tibetan Chicken fertilized full sib eggs were incubated up to Hamburger-Hamilton stage 43 under 13% and 21% oxygen concentration,respectively.Shouguang Chicken and Dwarf Recessive White Chicken were used as control groups.The hearts in all of the 3 chicken breeds under hypoxic and normoxic conditions were isolated and hybridized to Genechip Chicken Genome Array to study molecular mechanisms underlying the adaptation to high altitude of Tibetan Chicken.As a result,50 transcripts highly expressed in hypoxia are screened out.Among up-regulated genes,some are involved in the gene ontology(GO) such as cell growth,cell difference,muscle contraction and signal transduction.However,the expression levels of 21 transcripts are lower in hypoxia than those in normoxia.Some down-regulated genes take part in cell communication,ion transport,protein amino acid phosphorylation and signal transduction.Interestingly,gene enrichment analyses of these differential gene expressions are mainly associated with immune system response and ion channel activity in response to stimulus.Moreover,the transcriptional expression profiles analyzed by hierarchical clustering and CPP-SOM software in all of the 3 different chicken breeds revealed that Tibetan Chicken is much closely related to Shouguang Chicken rather than Dwarf Recessive White Chicken.In addition,12 transcripts of Tibetan Chicken breed-specific expressed genes were identified,which seem to result in a more effective and efficient induction of energy demand and signal transduction of transcription and suppression of abnormal development in response to hypoxia.These findings will be beneficial in clarifying the adaptive molecular mechanism of Tibetan Chicken as well as providing new insight into cardiovascular disease at high altitude medicine.

苗药组方熏蒸疗法对家兔早期膝骨性关节炎模型的差异基因分析及聚类分析

目的:探讨苗药组方熏蒸疗法对家兔早期膝骨性关节炎(knee osteoarthriti,KOA)模型差异表达基因进行层次聚类分析的意义。方法:将新西兰兔40只随机分为4组,空白对照组、模型对照组、苗药熏蒸组、清水对照组,每组10只。采用木瓜蛋白酶在兔膝关节腔内注射的方法复制早期KOA模型,造模成功后用苗药熏蒸疗法治疗20天,运用基因芯片检测技术对软骨组织的基因表达谱进行检测,最后将得到的原始数据通过RMA算法进行预处理,通过相关基因分析软件进行差异基因(difference gene,DEG)分析,并对其基因表达谱进行聚类分析。结果:DEG分析显示,模型对照组与空白对照组比较,差异显著基因有827个,上调435个,下调392个;苗药熏蒸组与模型对照组比较,差异显著基因有208个,上调112个,下调96个;清水熏蒸组与模型对照组比较,差异显著基因有152个,上调93个,下调59个。聚类分析显示,每个实验组的标本基本为一类,每个标本重复性好,每个实验组与对照组比较,它们的基因表达各有特点。结论:基因芯片检测可以发现苗药熏蒸疗法治疗早期KOA的差异表达基因,而聚类分析发现其相关基因的改变及其基因表达的特殊性。

生物信息学视角下抑郁及运动干预机制关键基因的研究进展

应激会导致抑郁,而运动对治疗抑郁是有益的,但这些作用的机制尚未被阐明。随着大数据时代的到来和生物信息技术的革新,通过比较基因的表达差异揭示疾病发生的分子机制逐渐成为高通量研究的常用策略。基因表达芯片可帮助研究者获取大量的抑郁相关基因信息,其中差异表达基因(differentially expressed genes,DEGs)是指抑郁或抑郁共病患者与正常人相比、治疗进行和运动干预前后,在转录组水平上表达有显著性差异的基因。综述研究发现,NDUFB9、CAMK2A、CACNA1D、NTRK2、CTSW、HES1、EGR1、NRN1、VEGF、GCC2等基因在抑郁发病机制、治疗机制、共病机制和运动干预机制中重复出现,且存在密切联系。生物信息分析结果显示,这些基因涉及能量代谢、氧化应激、表观遗传、炎症免疫、神经生长和发育、细胞生长存活与功能维持以及磷脂酰肌醇信号、Notch信号等相关功能和通路。

缺血性脑卒中差异表达基因筛选与生物信息学分析

目的 分析缺血性脑卒中(ischemic stroke,IS)基因表达芯片数据,筛选IS差异表达基因,利用生物信息分析其相关基因功能和信号通路,为IS发病机制提供分子理论基础。方法 从GEO数据库下载IS芯片表达数据,采用DESeq2、edgeR和limma包进行差异表达基因(differentially expressed genes,DEGs)的筛选。采用clusterProfiler包对DEGs进行GO功能和KEGG信号通路富集分析,应用STRING软件构建蛋白-蛋白互作(protein-protein interaction,PPI)网络,使用MCODE和cytoHubba插件筛选与IS发病密切相关的模块及枢纽基因。最后结合LASSO回归分析获取特征基因并利用ROC曲线评估其在IS中的诊断价值。结果 检索获得GSE22255、GSE58294和GSE16561 3个IS数据集,共包含195个外周血样本,合并表达矩阵获得包含89个IS病例和43个健康对照人群的新数据集,差异分析获得196个DEGs。GO功能和KEGG信号通路富集分析结果显示,上述基因共同介导应激反应、对刺激反应的调节等相关生物过程;参与沙门氏菌感染、NOD样受体和造血细胞谱系等的信号通路。PPI网络和Cytoscape分析获得排名前10的枢纽基因。LASSO回归分析获得11个特征基因与网络分析获得的10个枢纽基因交集,最终获得4个关键特征基因,分别是趋化因子受体7(CCR7)、基质金属蛋白酶9(MMP9)、Bcl-2相关蛋白A1(BCL2A1)和淋巴细胞抗原96(LY96)基因。ROC分析显示MMP9、BCL2A1和LY96基因可能是IS发病过程中的关键基因。结论 细胞凋亡、免疫和炎症过程等与IS的发生和发展密切相关。MMP9、BCL2A1和LY96关键基因是IS潜在的化疗和治疗靶点。

基于主成分分析的基因芯片数据研究

针对急性髓细胞白血病的基因芯片数据,通过筛选差异表达显著的基因,将原始数据划分为3个数据集并分别进行主成分分析,提取了3个主成分,计算主成分得分,获取第二、三主成分中得分绝对值靠前的基因作为关键基因。将3组数据集提取的关键基因进行对比,结果发现,HOXA9基因高频出现,说明HOXA9基因在AML中差异表达显著,该基因异常可能会导致AML的发生。

基于生物信息学分析类风湿性关节炎的潜在治疗靶点

目的 生物信息学分析类风湿性关节炎(RA)关键基因及信号通路。方法 检索GEO数据库中关于类风湿关节炎的相关芯片GSE77298,构建差异基因蛋白质-蛋白质相互作用关系(PPI),进行GO富集分析和KEGG通路分析,通过AnimalTFDB数据库预测转录因子。结果 GSE77298芯片中筛选出1 539个差异基因,其中有1 156个上调基因和383个下调基因;信号通路主要表现为类风湿性关节炎、金黄色葡萄球菌感染、趋化因子、病毒性心肌炎、细胞因子-细胞因子受体相互作用等;hub gene为CXCL12、CD44和CDH2;hsa-miR-30a-3p、hsa-miR-34a-5p、hsa-miR-30d-3p等10种主要miRNA;预测主要转录因子为GTF3C2、GLYR1、TRIM24、YY1等。结论 CXCL8、SOCS3、TLR2等可能是RA关键基因,has-miR-340-5p等10个重要miRNA可能参与RA发病过程,YY1等转录因子可能参与RA的疾病过程。

50 cGyγ射线照射人肝细胞基因差异表达谱研究

本研究利用基因芯片技术对50 cGyγ射线照射的人肝细胞和未照射的人肝细胞基因差异表达谱进行了研究和分析。研究结果表明:在所分析的14 000多条基因中,共有614条差异基因表达谱,其中明显上调的差异表达基因有521条,明显下调的差异表达基因93条。主要涉及到以下几种类型基因:与线粒体调控有关的基因;与甲型肝炎病毒受体有关的基因;与肿瘤坏死因子有关的基因;与细胞周期调控有关的基因;与激酶有关的基因;以及与锌指蛋白有关的基因等等。用RT-PCR进一步验证了部分差异表达基因:四个表达上调基因HAVcr-1、HAVcr-2、MFTC、MOAP1和2个表达下调基因TRIP12、DCN,检测结果与基因芯片结果相一致。

降低mRNA差异显示技术假阳性率的一种方法

为了探讨降低mRNA差异显示技术假阳性率的方法 ,进一步提高此技术的可靠性 ,提取了手术切除肝癌及非癌肝组织成对标本的总RNA ,逆转录获得cDNA片段 ,以mRNA差异显示方法筛选差异表达基因 ,选取较明显的一条差异表达条带 ,行进一步PCR扩增 .分别对PCR产物及其经TA克隆后随机挑选的 6个单克隆质粒DNA进行序列分析 ,并通过GenBank BLAST数据库进行序列的同源性比较 ,以Northern杂交予以来源确认 .自 72 0余条扩增条带中共选出 2 8条差异条带 .序列分析及同源性比较表明 ,所选择条带的PCR产物为一可能的新基因片段 ;而随机选择的 6个TA克隆质粒DNA中 ,有 4个为同一已知基因片段 ,一个为另一已知基因片段 ,一个为一可能的新基因片段 .同源性比较表明 ,PCR产物直接测序所得序列与TA克隆质粒DNA的 6个片段不具同源性 .结果表明 ,mRNA差异显示条带可能由 1条以上分子量相似的片段构成 ,直接对PCR产物行序列分析并以其为探针进行Northern杂交 ,是导致出现假阳性片段的原因之一 .将PCR产物进行TA克隆 ,对单克隆质粒DNA进行序列分析并以其为探针进行Northern杂交 ,可能是解决此问题的一种较好方法 .

脾气虚证患者基因差异表达研究

目的:研究慢性胃炎脾气虚证的特征性基因差异表达谱。方法:慢性胃炎脾气虚证患者及健康志愿者各选4例,镜下取胃黏膜组织,提取总RNA,一一配对制作基因芯片,采用荧光比值、生物信息学、双侧t检验等方法比较分析;实时荧光定量PCR检测相关基因。结果:差异表达基因54条,72.2%下调;其中与营养物质代谢和免疫调节相关差异表达基因45条,71.1%下调;差异基因中有4条基因P<0.05,为显著差异表达基因;实时定量PCR检测差异基因5条,4条与芯片结果一致。结论:慢性胃炎脾气虚证具有特征性的基因差异表达图谱,主要表现为与营养物质代谢及免疫调节相关基因呈下调趋势。

0.2 Gy γ射线照射人外周血淋巴细胞不同时间点差异基因表达谱研究

利用全基因组基因芯片技术对0.2Gyγ射线照后2、12、24小时点的人淋巴细胞和未照射的人淋巴细胞基因差异表达谱进行了研究和分析。结果表明,照后2小时的差异表达基因有44条,其中明显上调的差异表达基因有38条,明显下调的差异表达基因6条;照后12小时的差异表达基因有247条,其中明显上调的差异表达基因有151条,明显下调的差异表达基因96条;照后24小时的差异表达基因有704条差异基因表达谱,其中明显上调的差异表达基因有392条,明显下调的差异表达基因312条;共同差异表达基因只有6条。主要涉及到以下几类型基因:干扰素调节有关基因;生长抑制、DNA损伤有关基因;与P53调控有关的基因;与肿瘤坏死因子有关的基因;与细胞周期调控及凋亡有关的基因等等。RT-PCR结果表明,MERTK及TAIAP12个差异表达基因,其相对定量结果与基因芯片检验结果相一致。

应用cDNA微矩阵基因芯片筛选运动性心肌肥大相关基因的初步研究

目的 :应用cDNAchip(cDNA芯片或微矩阵基因芯片 )技术筛选运动性心肌肥大相关基因。方法 :摘取运动组和对照组小鼠心脏 ,按一步法抽提总RNA并纯化mRNA ,将 2 30 4个点包括10 3个阴性及对照基因和 2 2 0 1条小鼠靶基因的PCR扩增产物 ,按微矩阵 (12× 12点× 16亚矩阵 ,点间距离 375 μm)点制于硅烷化玻片上 ,制备成cDNA芯片。将等量抽提纯化的运动组和对照组小鼠心肌组织mRNA ,分别与掺入的荧光分子Cy3和Cy5经逆转录合成cDNA荧光探针 ,混合后再与cD NA芯片杂交 ,通过芯片扫描仪对荧光信号图像进行扫描 ,经计算机分析比较运动组和安静对照组心肌组织中基因表达谱的变化。结果 :在 2 2 0 1条待研究基因中 ,两组小鼠心肌组织间存在差异表达的基因。具有显著表达差异的基因有 71条 ,其中上调基因有 37条 ,下调基因有 34条。结果显示 :cDNA芯片技术筛选运动性心肌肥大相关基因具有高通量、高敏度、高效率、大规模和并行性等优点。

不同剂量γ射线照射人肝细胞差异基因表达谱的研究

利用基因芯片技术,对受不同剂量(0.5Gy、2Gy、4Gy)γ射线照射的人肝细胞和未受照射的人肝细胞基因差异表达谱进行了研究和分析。结果表明,在所分析的14000多条基因中,三个不同照射剂量共同差异表达的基因表达谱有284条,其中明显上调的差异表达基因有261条,明显下调的差异表达基因23条。主要涉及到以下几类型基因:与干扰素受体有关的基因;与线粒体调控有关的基因;与甲型肝炎病毒受体有关的基因;与细胞周期调控有关的基因;与激酶有关的基因;以及与锌指蛋白有关的基因等等。对4个表达上调基因HAVcr-1、HAVcr-2、MFTC、MOAP1和2个表达下调基因TRIP12和DCN进行了荧光定量RT-PCR验证,实验结果与基因芯片检验结果相一致。

γ射线照后不同时间点人肝细胞基因差异表达谱的研究

目的:对γ射线照射后4和24 h人肝细胞株的差异表达基因进行筛选,为从基因水平上揭示放射性从业人员肝脏的早期损伤提供依据。方法:利用全基因组基因芯片技术,对人正常肝细胞7702给予不同剂量(0.5和1.0 Gy)γ射线照射不同时间(4和24 h)后的基因差异表达谱进行筛选和分析。结果:照后4 h在不同剂量水平上(0.5和1.0 Gy)筛选出差异表达基因218个;照后24 h不同剂量水平上(0.5和1.0 Gy)筛选出差异表达基因1 475个;0.5 Gy剂量水平上照后不同时间(4和24 h)筛选出差异表达基因235个;1.0 Gy剂量水平上照后不同时间(4和24 h)筛选差异表达基因170个;最后筛选出共同的差异表达基因129个;另外还发现了一些有意义的通路途径,如胰岛素合成与分泌途径等;同时为了验证基因芯片筛选结果的准确性,进一步采用SYBR绿色实时荧光定量PCR技术,对胰岛素生长因子2结合蛋白3(IGF2BP3)及早期响应因子1(EGR1)两个有意义且表达量稳定的辐射后差异表达基因进行了验证,结果显示其定量结果与芯片检测结果趋势一致。结论:γ射线辐照后共筛选出差异表达基因129个,辐射对人肝细胞的早期损伤表现为多靶点、多层次及多通路等特点。

心经经脉与心脏相关的差异表达基因的研究

目的:从基因差异表达方面探讨心经与心脏相关的特异性基础。方法:复制大鼠急性心肌缺血模型,分别电针心经“神门”至“通里”段、小肠经“养老”至“支正”段、肺经“太渊”至“列缺”段。刺激电压5V,频率2Hz,波宽300μs,正负向交替方波,每次电刺激20min,1次/d,共3次。取缺血心肌组织,采用大鼠全基因U230序列芯片进行芯片检测,大规模筛选差异表达基因。结果:与模型组比较,差异表达2倍以上的基因,电针心经组有20个上调70个下调;电针小肠经组有18个上调26个下调;电针肺经组有14个上调20个下调,且3组之间鲜有相同的基因表达。结论:经脉脏腑相关具有确实的分子基础,电针心经、小肠经、肺经具有相对特异性作用途径。

水分胁迫条件下“洛旱2号”小麦根系的基因表达谱

为探讨小麦水分胁迫反应的分子调控机制,以抗旱小麦品种"洛旱2号"根系为材料,采用Affymetrix小麦基因芯片比较了20%PEG6000溶液处理后根系及对照的基因表达谱。结果表明,洛旱2号根系中受水分胁迫诱导而上调和下调表达的基因分别有3743个(4074个探针组)和4573个(5043个探针组),下调表达的基因远远多于上调表达的基因,其中上调2倍以上的1593个(1716个探针组),下调2倍以上的2238个(2451个探针组)。通过Affymetrix的Net Affx Analysis Center查询和NCBI的BLASTx分析,差异表达2倍以上的基因中有619个已注释了功能,利用MIPS数据库将注释基因分为10类,包括生物代谢、逆境胁迫反应、细胞组分生成、蛋白合成、细胞信号交流、细胞运输、转录相关、细胞分裂和蛋白质加工等。差异表达16倍以上的基因中有32个已注释了功能,其中与逆境胁迫反应相关的基因16个,与生物代谢相关的基因5个。利用实时定量RT-PCR对8个代表性候选基因在水分胁迫条件下的差异表达特性分析表明,其结果和芯片杂交分析的结果基本一致。