Functional analysis of differential microRNA expression profile in mouse fibrotic liver tissues induced by CCl4

AIM:To discover the expression profile of microRNAs (miRNAs) in mouse fibrotic liver tissues induced by carbon tetrachloride (CCl4), and to investigate the functions of these differential miRNAs based on the gene ontology (GO) analysis and KEGG Pathway analysis. METHODS:The mice were randomly divided into normal group and model group. Liver fibrosis was induced by subcutaneous injection of CCl4. miRNA expression profile of the liver tissues was assayed by a mouse miRNA microarray (Agilent 12.0). The differential expression of miRNAs between the normal and model mice was screened, and GO analysis and KEGG Pathway analysis were performed to determine the functions of these differential miRNAs. RESULTS:Thirty-nine miRNAs with differential expression were discovered in the model mice compared with the normal mice, among which 23 were up-regulated and 16 were down-regulated. GO analysis and KEGG Pathway analysis indicated that most pathological processes of liver fibrosis regulated by miRNAs included cell proliferation and activation, cell apoptosis, cell cycle, cell adhesion, inflammatory reaction, cell migration, transforming growth factor β (TGF-β) signaling pathway, Wnt signaling pathway and proteometabolism process. GO analysis revealed that the key up-regulated miRNAs were mmu-miR-322, mmu-miR-15b, mmu-miR-195, mmu-miR-200b and mmu-miR-214, and the key down-regulated miRNAs were mmu-miR-16, mmu-miR-130a, mmu-miR-101b, mmu-miR-30a and mmu-miR-30e. Analyzing the target genes screened out by GO analysis and Pathway analysis simultaneously, we found that the key up-regulated miRNAs included mmu-miR-200b, mmu-miR-322, mmu-miR-106b, mmu-miR-23a and mmu-miR-15b, and the key down-regulated miRNAs included mmu-miR-16, mmu-miR-30e, mmu-miR-30c, mmu-miR-30a and mmu-miR-130a. CONCLUSION: Differential expression of miRNAs is discovered in mouse fibrotic liver tissues induced by CCl4 compared with the normal liver tissues. Most of the pathological processes involved in liver fibrosis may be regulated by miRNA, such as cell proliferation and activation, cell adhesion and apoptosis, cell migration and differentiation, metabolism, TGF-β receptor signaling pathway and so on.     

Preliminary study on miRNA expression spectrum related to chemotherapy resistance in colon cancer

AIM: To screen the chemotherapy resistance-related microRNAs (miRNAs) of colon cancer using gene chip technique, and to explore the mechanism of miRNAs regulating chemotherapy resistance. METHODS: Gene chip technique was used to analyze the expression of miRNAs in colon cancer cell line HCT8 and vincristine-resistant cell line HCT8/v, and screen the miRNAs with significantly different expression. The results were verified by RT-qPCR. The target genes of these miRNAs were predicted, and the Gene Ontology (GO) analysis and the signaling pathway analysis of the predicted genes were carried out. RESULTS: Altogether 342 miRNAs with significantly differential expression were selected, in which 190 were up-regulated, and 152 were down-regulated. The verification results of RT-qPCR showed that the expression of miR-125-5p, miR-181c-5p and miR-153-3 was consistent with the results of chip detection. The expression of miR-130a-3p and miR-149-3p was not consistent with the results of chip detection. The results of GO analysis showed that the main pathway of chemotherapy resistance-related genes was RNA polymerase II regulatory region sequence-specific DNA binding. The chemotherapy resistance-related genes played roles mainly through positive regulation and are mainly located in intracellular membrane-bound organelles. The results of KEGG analysis showed that the pathways associated with the most enriched chemotherapy resistance-related genes were axon guidance pathway, insulin signaling pathway, and phospholipase D signaling pathway.CONCLUSION: miRNAs are closely related to chemotherapy resistance in colon cancer. Through the researches on miRNAs, we can have a deeper understanding of the mechanism of chemotherapy resistance and provide new ideas for reversing chemotherapy resistance in colon cancer.     

Differences of genome-wide methylation level and methylated genes between nasopharyngeal carcinoma cells in same genetic background but different radiation resistance

AIM: To compare the differences of the genome-wide methylation levels and methylated regions between nasopharyngeal carcinoma (NPC) cells in the same genetic background but different radiation resistance (CNE-2 cells and CNE-2R cells). METHODS: Using the method which was developed by Doctor Zhao Cun-you, based on using methyl-sensitive restriction enzyme to measure the genome-wide methylation levels. In addition, MeDIP-Seq was used to analyze the methylated regions in 6 gene functional elements, including the upstream 2k sequence, 5'UTR, coding sequence, intron, 3'UTR and downstream 2k sequence, between CNE-2 cells and CNE-2R cells. RESULTS: The genome-wide methylation level was approximately 30% lower in CNE-2R cells than that in CNE-2 cells. No obvious difference on the amount of genes and the coverage of the peak in the 6 gene functional elements was observed. However, the methylation pattern of plentiful genes had altered in the gene function elements. CONCLUSION: The genome-wide methylation levels and methylated regions between NPC cells in the same genetic background but different radiation resistance were quite different, indicating that the DNA methylation may be associated with NPC radioresistance.     

TMT-tagged quantitative proteomics analysis of differentially expressed proteins and enrichment of signaling pathways in nasopharyngeal carcinoma cells with or without SATB1 over-expression

AIM: To analyze the effects of special AT-rich sequence binding protein 1 (SATB1) expression on the protein expression profiles in human nasopharyngeal carcinoma (NPC) cells, and to enrich the differential signaling pathways through bioinformatics analysis. METHODS: SATB1 over-expressing lentivirus and negative control lentivirus were used to infect the CNE1 cells, and then the cell lines were obtained by puromycin stressed method. The total proteins of the 2 cells were extracted, and the differentially expressed proteins were screened by TMT-labeled protein quantification technique and tandem mass spectrometry. The mRNA levels of the differential protein-coding genes were verified by RT-qPCR. GO analysis was used to annotate and enrich the differentially expressed proteins, and the KEGG database was used to enrich and analyze the signaling pathways of differential proteins. RESULTS: SATB1 over-expressing CNE1 cells were established through infected with associated lentivirus. Compared with the control group, 278 differentially expressed proteins were identified in SATB1 over-expressing CNE1 cells, in which 115 were up-regulated and 163 were down-regulated. 10 representative differential protein-coding genes were verified by RT-qPCR, which showed the consistence with the proteomic results. GO analysis indicated differentially expressed proteins were mainly involved in cellular processes, single-organism processes, biological regulation, metabolic processes, protein binding and catalysis. Cell components of differentially expressed proteins mainly existed in cell part, cells and organelles. KEGG analysis showed that differentially expressed proteins were involved in signaling pathways closely related to tumors, includeing MAPK, PI3K-Akt, AMPK, JAK-STAT, p53, PPAR, Hippo and HIF-1 signaling pathways. CONCLUSION: Over-expression of SATB1 significantly alters the protein expression profiles in the NPC cells and affects multiple signaling pathways closely related to tumors. Proteomics also provides a possible macro approach to the screening of molecular mechanisms, therapeutic and prognostic targets for NPC.     

树体休眠期前苹果花芽对低温早期响应的转录组分析

【目的】从总体上了解苹果花芽早期响应低温信号的基因表达情况,以期了解苹果休眠期花芽早期反应的分子网络,从而为苹果抗冷性研究提供理论依据。【方法】于树体休眠前收集花芽,低温(4℃)处理45 min(T1)、90 min(T2)和240 min(T3),常温处理为对照(T0),利用转录组技术分析了树体休眠前苹果花芽响应低温信号早期的基因表达情况,利用实时荧光定量PCR(Quantitative Real-time PCR,qRT-PCR)进行数据验证。【结果】与对照相比,T1、T2和T3分别获得237、508和990个差异表达基因(Differentially expressed genes,DEGs)。GO富集分析表明:处理前期的DEGs主要涉及碳水化合物有关的代谢、单体碳水化合物代谢过程,而后期主要涉及刺激反应、胁迫响应和DNA的转录等生物学过程。KEGG富集分析表明DEGs主要参与了"植物-病原菌互作","植物激素信号转导"等。其中,在响应低温信号后,参与钙调素/钙调素类蛋白(Ca2+–CaM/CML)代谢的基因MDP0000808334、MDP0000263349等及参与脱落酸(Abscisic acid,ABA)、油菜素内酯(Brassinosteroid,BR)和赤霉素(Gibberellin,GA)信号代谢的基因MDP0000189486、MDP0000122792和MDP0000287039等上调表达显著。【结论】Ca2+信号通路可能主要参与了苹果花芽的冷响应过程。此外,ABA、BR和GA等激素可能在苹果花芽响应低温信号中也起重要的调控作用。     

虎奶菇菌丝体和菌核基因组甲基化分析

以虎奶菇[Pleurotus tuber-regium (Fr.) Singer]菌丝体和菌核为材料,利用MSAP技术分析其甲基化水平,并对甲基化差异片段进行回收测序,探究虎奶菇生长与DNA全基因组甲基化的关系.结果 表明,菌丝体和菌核的DNA甲基化率分别为7.94％和9.54％,其中半甲基化率分别为4.50％和4.0...     

Transcriptome profiling reveals differentially expressed genes associated with wizened flower bud formation in Chinese pear (Pyrus bretschneideri Rehd.)

Flower buds are very important for pear yield; non-germinated flower buds become wizened and drop from the branch, reducing pear production. However, little research has focused on the study of wizened flower bud (WB) formation in spring. In order to elucidate the mechanism of WB formation in pear, physiological indices relating to plant hormones and antioxidases were measured. We found that activities of peroxidase (POD) and superoxide dismutase (SOD) were higher and lower, respectively, in WBs than in normal flower buds (NBs). The contents of indole acetic acid (IAA), gibberellin (GA), and cytokinin (CTK) were lower in WBs, while the level of addition of abscisic acid (ABA) was higher in WBs than in NBs. Differentially expressed genes (DEGs) were surveyed between WBs and NBs. In total, 23 DEGs relating to POD and SOD were detected from GO (Gene Ontology) enrichment, and 29 DEGs associated with plant hormone biosynthesis were found from the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway. Notably, the expression patterns of these 52 genes were consistent with variations of antioxidase activities or hormone contents. Because POD and SOD are stress-response enzymes, the differences in POD and SOD activities between NBs and WBs indicated that WB formation in pear could result from ambient environmental stresses that influence expression levels of hormone biosynthesis genes.     

Gene microarray analysis of ovarian serous cystadenocarcinoma-related genes based on TCGA

AIM: To detect the differentially expressed genes associated with ovarian serous cystadenocarcinoma (OV) by microarray and to analyze the participated signaling pathway. METHODS: We analyzed 16 datasets of Affymetrix GeneChip Human Exon 1.0 ST Arrays from The Cancer Genome Atlas (TCGA), including 8 OV and 8 normal ovary samples. The function of differential genes was determined by pathway and gene ontology (GO) analysis. The probable functions of the key genes were predicted according to intergenic signal transduction network. RESULTS: The 1 144 genes were identified as distinctively expressed in OV (P<0.05), 747 of which were up-regulated and 397 were down-regulated. The GO analysis results showed that the altered genes were involved in 362 up-regulated and 160 down-regulated significant functions (P<0.05) related to cell cycle, DNA replication, cell proliferation, cell apoptosis, cell adhesion, etc. The pathways of the different genes were involved in the 59 enrichment-related pathways (P<0.05), 45 of which were up-regulated and 14 were down-regulated. Among the 59 pathways, cell cycle, P53 signaling pathway, DNA replication, pathways in cancer, PI3K-Akt signaling pathway, ECM-receptor signaling pathway, cell adhesion molecules and cell apoptosis were related to tumor genesis, development and metastasis. As a result, 229 genes with significant functions and pathways in GO and pathway analysis were selected to construct signal transduction network (Signal-Net), 4 of which, CDK1, PLK1, MCM3 and PGK1, were found to play key roles in OV signal regulation network. CONCLUSION: The OV shows abundant differentially expressed genes that play key roles in cancer-related signal pathways.     

结球甘蓝自花和异花授粉诱导的柱头转录组分析

分别以自花授粉30 min、异花授粉30 min和未授粉处理的甘蓝柱头进行Illumina HiSeq2000高通量转录组测序,分析自交不亲和性甘蓝自花授粉、异花授粉柱头与未授粉柱头中基因表达的差异。转录组测序共获得1.6 × 108对原始序列读取片段,总碱基数为2.38 × 1010 bp,与未授粉对照相比,自花、异花授粉后差异表达的基因分别有2 900个和2 328个,共有的差异表达基因1 904个。在差异表达基因背景和全部基因背景下,自花授粉较大比例的差异基因是细胞杀伤、胞外基质部分和核酸结合转录因子活性;异花授粉较大比例的差异基因是信号传递、胞外区域部分、结构分子活性和营养库活性。GO功能的显著性富集分析表明,自花授粉处理后特有的40个极显著的富集条目主要涉及糖跨膜转运活性、微管结合和钙调素依赖性蛋白激酶活性等。异花授粉特有的53个极显著的富集条目主要涉及法尼酸O–甲基转移酶的活性、微管负向运动和生长素跨膜运输等。自花授粉处理后特异差异表达的996个基因主要涉及钙离子结合、细胞骨架相关、茉莉酸和水杨酸代谢等生物过程,异花授粉处理后特异差异表达的424个基因主要涉及物质跨膜转运及脂质代谢。     

白榆二倍体及同源四倍体的生理特性及转录组差异分析

为了探究白榆二倍体及其同源四倍体的基因表达谱差异情况,以白榆二倍体及其同源四倍体为材料,比较二者成熟叶片的生理特征并采摘幼嫩叶片进行转录组数据分析。结果显示,与二倍体相比,同源四倍体丙二醛含量、可溶性蛋白含量及叶绿素含量增加,可溶性糖含量降低。转录组测序结果共得到2 407个差异表达基因,其中上调基因为1 076个,下调基因为1 331个。在COG蛋白质的同源注释分析得到918个差异表达基因,主要涉及21个方面。共有1 380个差异表达基因被GO注释,主要与细胞组分、代谢功能、催化活性等相关。通过KEGG通路注释发现,共有930个差异表达基因分布到5大类KEGG通路中,其中以代谢机制中差异表达基因被注释到的最多。叶绿素含量的增加使四倍体的叶片颜色变得更加浓绿,丙二醛、可溶性糖、可溶性蛋白的含量的增加与减少与四倍体植物在植物代谢与生长上有一定关系,经过转录组测序分析,糖酵解途径的基因表达为下调,还原性戊糖磷酸循环与光呼吸等与叶绿体有关的基因为上调。     

Aberrant DNA methylation in promoter region of p53 gene in acute leukemia

AIM: To investigate the significance of aberrant p53 gene promoter methylation in acute leukemia by detecting the occurrence of p53 gene promoter methylation. METHODS: Genomic DNA was digested using restriction endonuclease MspⅠ, HpaⅡ, EcoRⅡ, BstNⅠ, respectively. PCR amplification was conducted and the products after digestion and genomic DNA were used as template. The PCR product was subjected to electrophoresis and the results were analyzed by gel imaging and analysis system. Parts of the separated DNA were sequenced after purification from gel. RESULTS: The prevalence of methylation in acute leukemia group was 38.7％, of which ALL was 45.5％ (5 of 11) and ANLL 35.0％ (7 of 20). No methylation was detected in normal control group. There was significant difference between the prevalence of methylation in acute leukemia group and the normal control group (Fisher′s exact test, P<0.05). However, the prevalence between ALL and ANLL was not significantly different (Fisher′s exact test, P>0.05). Compared the relationship between aberrant methylation of p53 gene and clinical data, statistical significance between aberrant methylation of p53 gene and enlargement of lymph nodes, liver or spleen(P<0.05) was observed. CONCLUSION: ①Aberrant DNA methylation in P1 promoter region of p53 gene exits in part of acute leukemic patients, but not in health people. ②The prevalence of aberrant DNA methylation between ALL and ANLL is not significantly different. ③The patients with aberrant methylation of p53 gene seem to show more frequently the manifestations of enlarged lymph nodes, liver or spleen than usual.     

Progress of embryonic stem cells during directional differentiation regulated by epigenetic modification

Embryonic stem cells undergo extensive self-renewal and have the capacity to differentiate along multiple cell lineages. Research on totipotency and directional differentiation of embryonic stem cells in order to treat intractable disease, such as cancer, heart failure, atherosclerosis by tissue regeneration and cell transplantation are investigated. Epigenetic modification, including DNA methylation, chromatin restructure, and non-coding RNA-mediated regulatory events, regulate the differentiation of embryonic stem cells without detectable genetic changes. These mechanisms are often associated with starting-up and maintenance of epigenetic silence. The achievement and focuses on the molecular mechanism of embryonic stem cells during directional differentiation regulated by epigenetic modification are reviewed.     

金针菇单核体DAN3和M与其杂交双核体G1菌丝阶段基因表达差异的转录组初步分析

以金针菇(Flammulinavelutipes)单核菌株DAN3的基因组为参考,完成单核体DAN3和M及其杂交双核体G1在菌丝阶段的转录组测序与数据分析,比较两个样本间的差异基因,并对差异基因进行GO功能分析和KEGG pathway分析.结果表明:两个样本中共有显著性差异表达的基因86个,其中,在G1中呈上调、下调表达的基因数分别为41、45个,有2个基因在G1中特异表达.GO功能分析结果表明,86个差异基因中有40个基因比对上了GO功能注释,其中18个基因在G1中呈上调表达;单一生物过程和催化活性为显著性富集的功能,相关基因在G1中呈上调表达.KEGG pathway分析结果表明,22个差异基因被定位到17条Pathway,其中10个基因在G1中呈上调表达,包括赖氨酸代谢途径对应基因,3_M和G1菌丝样品中赖氨酸含量分别为1.70×103 ng/mg和1.06×103 ng/mg,说明G1中上调的基因可能与之降解相关;DNA复制是显著性富集的代谢途径,相关基因在G1中呈下调表达.     

基于转录组水平的枣疯病发病机理研究

为探究枣树感染枣疯病植原体后的发病规律和内在机制,以‘木枣’健康与感病植株为材料,连续测定现蕾后10~60 d叶片中玉米素、生长素、脱落酸、赤霉素、水杨酸等内源激素的含量以及过氧化物酶、超氧化物歧化酶、过氧化氢酶等抗氧化保护酶的活性;通过转录组测序,筛选感病诱发的差异表达基因,并利用qRT-PCR技术对相关基因的表达模式进行验证。结果表明:感病植株中玉米素含量在现蕾后30 d时开始显著升高,水杨酸含量在现蕾后50 d时开始显著升高,过氧化氢酶活性呈降低趋势但在现蕾后60 d时显著升高。以现蕾后20~30 d的叶片进行转录组测序分析,共筛选到1 669个差异表达基因,其中1 114个基因在感病植株中上调表达,555个下调表达。差异表达基因的GO功能富集主要包括生物进程、代谢进程及催化活性,KEGG代谢通路主要为次级生物代谢。在差异表达基因中发现了15个与激素和保护酶代谢相关的基因,经qRT-PCR验证,ZjNCED、ZjGA20、ZjPAL、ZjPOD2和ZjPOD5在转录水平上的表达与对应的激素含量和保护酶活性变化趋势一致,表明这些基因的表达调控对感病后植株的内源激素含量和抗氧化保护酶活性的动态变化具有重要作用。枣疯病植原体感染枣树引起基因表达改变、激素和保护酶的代谢紊乱可能是导致枣树发育畸形的原因。