BACKGROUND Hepatocellular carcinoma(HCC)is the most common subtype of primary liver cancer with varied incidence and epidemiology worldwide.Sorafenib is still a recommended treatment for a large proportion of patients...BACKGROUND Hepatocellular carcinoma(HCC)is the most common subtype of primary liver cancer with varied incidence and epidemiology worldwide.Sorafenib is still a recommended treatment for a large proportion of patients with advanced HCC.Different patterns of treatment responsiveness have been identified in differentiated hepatoblastoma HepG2 cells and metastatic HCC SNU449 cells.AIM To define the long non-codingRNA-microRNA-mRNA(lncRNA-miRNA-mRNA)predicted signatures related to selected hallmarks of cancer(apoptosis,autophagy,cell stress,cell dedifferentiation and invasiveness)in RNAseq studies using Sorafenib-treated HepG2 and SNU449 cells.Various available software analyses allowed us to establish the lncRNA-miRNA-mRNA regulatory axes following treatment in HepG2 and SNU449 cells.METHODS HepG2 and SNU449 cells were treated with Sorafenib(10μmol/L)for 24 hours.Total RNA,including small and long RNA,was extracted with a commercial miRNeasy kit.RNAseq was carried out for the identification of changes in lncRNA-miRNA-mRNA regulatory axes.RESULTS MALAT,THAP9-AS1 and SNGH17 appeared to coordinately regulate miR-374b-3p and miR-769-5p that led to upregulation of SMAD7,TIRARP,TFAP4 and FAXDC2 in HepG2 cells.SNHG12,EPB41 L4A-AS1,LINC01578,SNHG12 and GAS5 interacted with let-7b-3p,miR-195-5p and VEGFA in SNU449 cells.The axes MALAT1/hsamir-374b-3p/SMAD7 and MALAT1/hsa-mir-769-5p/TFAP4 were of high relevance for Sorafenib response in HepG2 cells,whereas PVT1/hsa-miR-195-5p/VEGFA was responsible for the differential response of SNU449 cells to Sorafenib treatment.CONCLUSION Critical lncRNAs acting as sponges of miRNA were identified that regulated mRNA expression,whose proteins mainly increased the antitumor effectiveness of the treatment(SMAD7,TIRARP,TFAP4,FAXDC2 and ADRB2).However,the broad regulatory axis leading to increased VEGFA expression may be related to the side effect of Sorafenib in SNU449 cells.展开更多
An accurate assessment of host and pathogen gene expression during infection is critical for understanding the molecular aspects of host-pathogen interactions.Often,pathogen-derived transcripts are difficult to ascert...An accurate assessment of host and pathogen gene expression during infection is critical for understanding the molecular aspects of host-pathogen interactions.Often,pathogen-derived transcripts are difficult to ascertain at early infection stages owing to the unfavourable transcript representation compared to the host genes.In this study,we compare two sequencing techniques,RNAseq and enrichment sequencing(RenSeq and PenSeq)of cDNA,to investigate gene expression patterns in the doubled monoploid potato(DM)infected with the late blight pathogen Phytophthora infestans.Our results reveal distinct advantages of cDNA RenSeq and PenSeq over traditional RNAseq in terms of target gene representation and transcriptional quantification at early infection stages.Throughout the infection time course,cDNA enrichment sequencing enables transcriptomic analyses for more targeted host and pathogen genes.For highly expressed genes that were sampled in parallel by both cDNA enrichment and RNAseq,a high level of concordance in expression profiles is observed,indicative of at least semi-quantitative gene expression representation following enrichment.展开更多
Inflammation plays a crucial role in the regeneration of fish and avian retinas.However,how inflammation regulates Müller glia(MG)reprogramming remains unclear.Here,we used single-cell RNA sequencing to investiga...Inflammation plays a crucial role in the regeneration of fish and avian retinas.However,how inflammation regulates Müller glia(MG)reprogramming remains unclear.Here,we used single-cell RNA sequencing to investigate the cell heterogeneity and interactions of MG and immune cells in the regenerating zebrafish retina.We first showed that two types of quiescent MG(resting MG1 and MG2)reside in the uninjured retina.Following retinal injury,resting MG1 transitioned into an activated state expressing known reprogramming genes,while resting MG2 gave rise to rod progenitors.We further showed that retinal microglia can be categorized into three subtypes(microglia-1,microglia-2,and proliferative)and pseudotime analysis demonstrated dynamic changes in microglial status following retinal injury.Analysis of cell–cell interactions indicated extensive crosstalk between immune cells and MG,with many interactions shared among different immune cell types.Finally,we showed that inflammation activated Jak1–Stat3 signaling in MG,promoting their transition from a resting to an activated state.Our study reveals the cell heterogeneity and crosstalk of immune cells and MG in zebrafish retinal repair,and may provide valuable insights into future mammalian retina regeneration.展开更多
Background The pituitary belongs to the most important endocrine glands involved in regulating reproductive functions.The proper functioning of this gland ensures the undisturbed course of the oestrous cycle and affec...Background The pituitary belongs to the most important endocrine glands involved in regulating reproductive functions.The proper functioning of this gland ensures the undisturbed course of the oestrous cycle and affects the female's reproductive potential.It is believed that visfatin,a hormone belonging to the adipokine family,may regulate reproductive functions in response to the female's metabolic state.Herein we ve rified the hypothesis that suggests a modulatory effect of visfatin on the anterior pituitary transcriptome during the mid-luteal phase of the oestrous cycle.Results RNA-seq analysis of the porcine anterior pituitary cells revealed changes in the expression of 202 genes(95 up-regulated and 107 down-regulated in the presence of visfatin,when compared to the non-treated controls),assigned to 318 gene ontology terms.We revealed changes in the frequency of alternative splicing events(235 cases),as well as long noncoding RNA expression(79 cases)in the presence of the adipokine.The identified genes were associated,among others,with reproductive system development,epithelial cell proliferation,positive regulation of cell development,gland morphogenesis and cell chemotaxis.Conclusions The obtained results indicate a modulatory influence of visfatin on the regulation of the porcine transcriptome and,in consequence,pituitary physiology during the mid-luteal phase of the oestrous cycle.展开更多
目的利用二代测序技术筛选出具有显著性变化的基因,并探讨月腺大戟素A在转录组学层面上抗乳腺癌活性的作用机制。方法从月腺大戟药材中提取乙酰间苯三酚类化合物月腺大戟素A,对MCF-7细胞(乳腺癌细胞的luminal A型)进行干扰,观察被干扰...目的利用二代测序技术筛选出具有显著性变化的基因,并探讨月腺大戟素A在转录组学层面上抗乳腺癌活性的作用机制。方法从月腺大戟药材中提取乙酰间苯三酚类化合物月腺大戟素A,对MCF-7细胞(乳腺癌细胞的luminal A型)进行干扰,观察被干扰后的细胞与正常细胞差异基因表达,采用二代高通量测序平台(IlluminaHi-Seq)测序技术分别对对照组和实验组各3个样本进行高通量转录组测序并进行数据分析。结果对照组和实验组分别总获得123656848、123974262个干净序列(clean reads),分别对比到参考基因组上的序列为119762214、119881622,各占总数的96.85%、96.69%;2组转录组对照可得:差异基因总数为1695个,其中上调基因770个,下调基因925个,可清楚注释的基因有3874个。应用基因本体论(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)进行生物功能富集分析,GO分析发现这3874个基因主要涉及生物过程(1270个)、细胞组成(1322个)与分子功能(1282个)3个大类的45个小类,包括细胞的生长发育过程、信号蛋白活性、膜以及基因表达的调控等过程;KEGG分析发现差异表达基因涉及263条信号通路,主要代谢通路为PI3K-Akt信号通路、MAPK信号通路;以及碳水化合物代谢、心肌系统和细胞生殖系统等生物过程。结论利用二代高通量测序平台测序技术一共筛选、鉴定出差异基因1695个,更深入了解了月腺大戟素A与MCF-7细胞基因之间的相互关系,为乳腺癌治疗提供了一些理论基础。展开更多
RNA sequencing(RNAseq)can reveal gene fusions,splicing variants,mutations/indels in addition to differential gene expression,thus providing a more complete genetic picture than DNA sequencing.This most widely used tec...RNA sequencing(RNAseq)can reveal gene fusions,splicing variants,mutations/indels in addition to differential gene expression,thus providing a more complete genetic picture than DNA sequencing.This most widely used technology in genomics tool box has evolved from classic bulk RNA sequencing(RNAseq),popular single cell RNA sequencing(scRNAseq)to newly emerged spatial RNA sequencing(spRNAseq).Bulk RNAseq studies average global gene expression,scRNAseq investigates single cell RNA biology up to 20,000 individual cells simultaneously,while spRNAseq has ability to dissect RNA activities spatially,representing next generation of RNA sequencing.This article highlights these technologies,characteristic features and suitable applications in precision oncology.展开更多
Polyploid plants often exhibit enhanced stress tolerance relative to their diploid counterparts,but the physiological and molecular mechanisms of this enhanced stress tolerance remain largely unknown.In this study,we ...Polyploid plants often exhibit enhanced stress tolerance relative to their diploid counterparts,but the physiological and molecular mechanisms of this enhanced stress tolerance remain largely unknown.In this study,we showed that autotetraploid trifoliate orange(Poncirus trifoliata(L.)Raf.)exhibited enhanced salt tolerance in comparison with diploid progenitors.Global transcriptome profiling of diploid and tetraploid plants with or without salt stress by RNAseq revealed that the autotetraploids displayed specific enrichment of differentially expressed genes.Interestingly,the leaves and roots of tetraploids exhibited different expression patterns of a variety of upregulated genes.Genes related to plant hormone signal transduction were enriched in tetraploid leaves,whereas those associated with starch and sucrose metabolism and proline biosynthesis were enriched in roots.In addition,genes encoding different antioxidant enzymes were upregulated in the leaves(POD)and roots(APX)of tetraploids under salt stress.Consistently,the tetraploids accumulated higher levels of soluble sugars and proline but less ROS under salt stress compared to the diploids.Moreover,several genes encoding transcription factors were induced specifically or to higher levels in the tetraploids under salt stress.Collectively,this study demonstrates that the activation of various multifaceted defense systems in leaves and roots contributes to the enhanced salt tolerance of autotetraploids.展开更多
Taking advantage of the fast-growing knowledge of RNA-binding proteins(RBPs)we review the signature of downregulated genes for RBPs in the transcriptome of induced pluripotent stem cell neurons(iNeurons)modelling the ...Taking advantage of the fast-growing knowledge of RNA-binding proteins(RBPs)we review the signature of downregulated genes for RBPs in the transcriptome of induced pluripotent stem cell neurons(iNeurons)modelling the neurodevelopmental Rubinstein Taybi Syndrome(RSTS)caused by mutations in the genes encoding CBP/p300 acetyltransferases.We discuss top and functionally connected downregulated genes sorted to“RNA processing”and“Ribonucleoprotein complex biogenesis”Gene Ontology clusters.The first set of downregulated RBPs includes members of hnRNHP(A1,A2B1,D,G,H2-H1,MAGOHB,PAPBC),core subunits of U small nuclear ribonucleoproteins and Serine-Arginine splicing regulators families,acting in precursor messenger RNA alternative splicing and processing.Consistent with literature findings on reduced transcript levels of serine/arginine repetitive matrix 4(SRRM4)protein,the main regulator of the neural-specific microexons splicing program upon depletion of Ep300 and Crebbp in mouse neurons,RSTS iNeurons show downregulated genes for proteins impacting this network.We link downregulated genes to neurological disorders including the new HNRNPH1-related intellectual disability syndrome with clinical overlap to RSTS.The set of downregulated genes for Ribosome biogenesis includes several components of ribosomal subunits and nucleolar proteins,such NOP58 and fibrillarin that form complexes with snoRNAs with a central role in guiding post-transcriptional modifications needed for rRNA maturation.These nucleolar proteins are“dual”players as fibrillarin is also required for epigenetic regulation of ribosomal genes and conversely NOP58-associated snoRNA levels are under the control of NOP58 interactor BMAL1,a transcriptional regulator of the circadian rhythm.Additional downregulated genes for“dual specificity”RBPs such as RUVBL1 and METTL1 highlight the links between chromatin and the RBP-ome and the contribution of perturbations in their cross-talk to RSTS.We underline the hub position of CBP/p300 in chromatin regulation,the impact of its defect on neurons’post-transcriptional regulation of gene expression and the potential use of epidrugs in therapeutics of RBP-caused neurodevelopmental disorders.展开更多
Influenza viruses not only cause respiratory illness,but also have been reported to elicit neurological manifestations following acute viral infection.The central nervous system(CNS)has a specific defense mechanism ag...Influenza viruses not only cause respiratory illness,but also have been reported to elicit neurological manifestations following acute viral infection.The central nervous system(CNS)has a specific defense mechanism against pathogens structured by cerebral microvasculature lined with brain endothelial cells to form the blood–brain barrier(BBB).To investigate the response of human brain microvascular endothelial cells(hBMECs)to the Influenza A virus(IAV),we inoculated the cells with the A/WSN/33(H1N1)virus.We then conducted an RNAseq experiment to determine the changes in gene expression levels and the activated disease pathways following infection.The analysis revealed an effective activation of the innate immune defense by inducing the pattern recognition receptors(PRRs).Along with the production of proinflammatory cytokines,we detected an upregulation of interferons and interferon-stimulated genes,such as IFN-β/λ,ISG15,CXCL11,CXCL3 and IL-6,etc.Moreover,infected hBMECs exhibited a disruption in the cytoskeletal structure both on the transcriptomic and cytological levels.The RNAseq analysis showed different pathways and candidate genes associated with the neuroactive ligand-receptor interaction,neuroinflammation,and neurodegenerative diseases,together with a predicted activation of the neuroglia.Likewise,some genes linked with the mitochondrial structure and function displayed a significantly altered expression.En masse,this data supports that hBMECs could be infected by the IAV,which induces the innate and inflammatory immune response.The results suggest that the influenza virus infection could potentially induce a subsequent aggravation of neurological disorders.展开更多
Background:To treat vascular proliferative diseases,anti-VEGF therapies have shown systemic adverse effects attributable to the lack of selectivity between pathological and physiological angiogenesis.Thus,identifying ...Background:To treat vascular proliferative diseases,anti-VEGF therapies have shown systemic adverse effects attributable to the lack of selectivity between pathological and physiological angiogenesis.Thus,identifying the molecular mechanisms that are only specific to pathological cell types is crucial to develop better precision medicine.Methods:Here,we used different cell type enrichment approaches combined with single-cell RNA sequencing to define the transcriptomic changes within each retinal cell types in a mouse model of ischemic retinopathy.This retinal model develops pathological neovascularization(NV)in response to local hypoxia following oxygen-induced vessel obliteration(P7 to P12).The NV phenotype is characterized by the progressive appearance of vascular tufts resulting from misguided,abnormal proliferation of endothelial cells that we monitored at 3 consecutive time points-P12,P14 and P17(peak of NV).Results:By following the dynamic response to hypoxia,our experimental design reveals how pathological angiogenesis is specifically associated with significant metabolic adaptations in different subtypes of endothelial cells(i.e.,Tips vs Stalk cells).We also identify a pathological subtype of glial cells over-expressing VEGFA and pro-inflammatory IL-1 receptor subunits.This subtype of activated glial cells was targeted using selective IL1R antagonist treatment which reduced glial activation,inflammation,NV and promotes physiological angiogenesis,therefore improving tissue regeneration.Conclusions:Our results illustrate how analyzing cell type heterogeneity in tissues developing pathological angiogenesis allows establishing better targeting therapies to restore vascular integrity.展开更多
目的分析比较不同肿瘤基质评分胃癌患者的基因表达特征,鉴定与评分相关的胃癌预后基因,以期为临床胃癌诊断和预后提供更精准的手段。方法从癌症基因组图谱数据库(the cancer genome atals,TCGA)下载胃癌的临床资料和组织转录组测序(ribo...目的分析比较不同肿瘤基质评分胃癌患者的基因表达特征,鉴定与评分相关的胃癌预后基因,以期为临床胃癌诊断和预后提供更精准的手段。方法从癌症基因组图谱数据库(the cancer genome atals,TCGA)下载胃癌的临床资料和组织转录组测序(ribonucleic acid sequencing,RNAseq)表达数据。从基质免疫评估数据库(estimation of stromal and immune cells in malignant tumor tissues using expression data,ESTIMATE)网站下载TCGA数据库中胃癌患者基质评分信息。获取患者的临床信息、RNAseq表达谱、基质评分。按照基质评分的高低分为高基质评分组和低基质评分组,分析基质评分与胃癌预后的关系。用R语言DEseq2包进行标准化处理和差异分析;WGCNA(weight correlation network analysis,WGCNA)包筛选与基质评分密切相关的差异基因;单因素COX风险比例回归模型(COX proportional model,COX)初步筛选基质评分密切相关基因中与胃癌预后相关的基因;LASSO(least absolute shrinkage and selection operator,LASSO)回归模型筛选出其中影响胃癌预后的关键基因,计算最小λ值;多因素COX回归分析构建关键基因胃癌预后模型,并量化基因表达量与患者生存时间的关系;模型内部绘制关键基因的生存曲线。最后通过其他公共数据库(KM-plotter数据库和Oncomine数据库)验证这些基因在胃癌大样本的表达和预后。结果基质评分越高的患者表现为预后更差(P<0.05)。对患者的RNA-seq差异表达分析筛选得到1581个差异表达基因;从中通过WGCNA筛选出1015个基因与胃癌基质评分密切相关;单因素COX回归选出377个基因与胃癌患者预后相关(P<0.05);LASSO回归筛选出12个与胃癌预后相关的关键基因,最小λ=12;多因素COX回归分析显示该模型C指数为0.68,3年生存期和5年生存期的预测值基本贴合实际值,3年生存期曲线下面积(area under the curve,AUC)为0.693,5年生存时间AUC为0.725。12个基因中,ACTA1、ADAMTS12、LINCO1614、MATN3、MTUS2、PLCL1、POSTN、SERPINE1、TPTEP1表达量越高,患者生存期越短,GAD1和MMP16表达量的越低,患者生存期越短;6个基因(ADAMTS12、MATN3、MEGF10、PLCL1、POSTN、SERPINE)各自作为独立危险因素,具有最佳的胃癌预后预测功能(P<0.05)。KM-plotter数据库和Oncomine数据库符合本研究的预测结果。结论肿瘤基质评分越高的胃癌患者,预后更差、生存周期更短。6个基因ADAMTS12、MEGF10、PLCL1、POSTN、MATN3、SERPINE与患者的肿瘤基质评分及预后密切相关。其表达越高,患者评分越高,预后越差、生存周期越短。本研究鉴定了与胃癌基质评分相匹配的预后基因,提示胃癌基质研究的进一步方向。展开更多
基金Supported by Instituto de Salud Carlos III(ISCiii),No.PI19/01266 and No.PI22/00857Consejería de Salud y Familias(Junta de Andalucía),No.PI-0216-2020 and No.PIP-0215-2020Biomedical Research Network Center for Liver and Digestive Diseases(CIBERehd)founded by the ISCIII and co-financed by European Regional Development Fund“A way to achieve Europe”ERDF.
文摘BACKGROUND Hepatocellular carcinoma(HCC)is the most common subtype of primary liver cancer with varied incidence and epidemiology worldwide.Sorafenib is still a recommended treatment for a large proportion of patients with advanced HCC.Different patterns of treatment responsiveness have been identified in differentiated hepatoblastoma HepG2 cells and metastatic HCC SNU449 cells.AIM To define the long non-codingRNA-microRNA-mRNA(lncRNA-miRNA-mRNA)predicted signatures related to selected hallmarks of cancer(apoptosis,autophagy,cell stress,cell dedifferentiation and invasiveness)in RNAseq studies using Sorafenib-treated HepG2 and SNU449 cells.Various available software analyses allowed us to establish the lncRNA-miRNA-mRNA regulatory axes following treatment in HepG2 and SNU449 cells.METHODS HepG2 and SNU449 cells were treated with Sorafenib(10μmol/L)for 24 hours.Total RNA,including small and long RNA,was extracted with a commercial miRNeasy kit.RNAseq was carried out for the identification of changes in lncRNA-miRNA-mRNA regulatory axes.RESULTS MALAT,THAP9-AS1 and SNGH17 appeared to coordinately regulate miR-374b-3p and miR-769-5p that led to upregulation of SMAD7,TIRARP,TFAP4 and FAXDC2 in HepG2 cells.SNHG12,EPB41 L4A-AS1,LINC01578,SNHG12 and GAS5 interacted with let-7b-3p,miR-195-5p and VEGFA in SNU449 cells.The axes MALAT1/hsamir-374b-3p/SMAD7 and MALAT1/hsa-mir-769-5p/TFAP4 were of high relevance for Sorafenib response in HepG2 cells,whereas PVT1/hsa-miR-195-5p/VEGFA was responsible for the differential response of SNU449 cells to Sorafenib treatment.CONCLUSION Critical lncRNAs acting as sponges of miRNA were identified that regulated mRNA expression,whose proteins mainly increased the antitumor effectiveness of the treatment(SMAD7,TIRARP,TFAP4,FAXDC2 and ADRB2).However,the broad regulatory axis leading to increased VEGFA expression may be related to the side effect of Sorafenib in SNU449 cells.
基金supported by the Rural & Environment Science & Analytical Services (RESAS) Division of the Scottish Government through project JHI-B1-1the Biotechnology and Biological Sciences Research Council (BBSRC) through awards BB/ S015663/1+2 种基金BB/X009068/1Research Leaders 2025 fellowship funded by European Union’s Horizon 2020 research and innovation programme under Marie Sklodowska-Curie grant agreement no. 754380the Research/Scientific Computing teams at The James Hutton Institute and NIAB for providing computational resources and technical support for the “UK’s Crop Diversity Bioinformatics HPC” (BBSRC grant BB/ S019669/1)。
文摘An accurate assessment of host and pathogen gene expression during infection is critical for understanding the molecular aspects of host-pathogen interactions.Often,pathogen-derived transcripts are difficult to ascertain at early infection stages owing to the unfavourable transcript representation compared to the host genes.In this study,we compare two sequencing techniques,RNAseq and enrichment sequencing(RenSeq and PenSeq)of cDNA,to investigate gene expression patterns in the doubled monoploid potato(DM)infected with the late blight pathogen Phytophthora infestans.Our results reveal distinct advantages of cDNA RenSeq and PenSeq over traditional RNAseq in terms of target gene representation and transcriptional quantification at early infection stages.Throughout the infection time course,cDNA enrichment sequencing enables transcriptomic analyses for more targeted host and pathogen genes.For highly expressed genes that were sampled in parallel by both cDNA enrichment and RNAseq,a high level of concordance in expression profiles is observed,indicative of at least semi-quantitative gene expression representation following enrichment.
基金supported by the National Natural Science Foundation of China,Nos.81970820(to HX),31771644(to JL),31930068(to JL),82371176(to JL),81801331(to LC)National Key Research and Development Project of China.Nos.2017YFA0104100(to JL),2017YFA0701304(to HX)+1 种基金Shanghai Yangzhi Rehabilitation Hospital(Shanghai Sunshine Rehabilitation Center)Talent Introduction Plan,No.KYPT202204(to LC)the Fundamental Research Funds for the Central Universities,No.22120230292(to JL)。
文摘Inflammation plays a crucial role in the regeneration of fish and avian retinas.However,how inflammation regulates Müller glia(MG)reprogramming remains unclear.Here,we used single-cell RNA sequencing to investigate the cell heterogeneity and interactions of MG and immune cells in the regenerating zebrafish retina.We first showed that two types of quiescent MG(resting MG1 and MG2)reside in the uninjured retina.Following retinal injury,resting MG1 transitioned into an activated state expressing known reprogramming genes,while resting MG2 gave rise to rod progenitors.We further showed that retinal microglia can be categorized into three subtypes(microglia-1,microglia-2,and proliferative)and pseudotime analysis demonstrated dynamic changes in microglial status following retinal injury.Analysis of cell–cell interactions indicated extensive crosstalk between immune cells and MG,with many interactions shared among different immune cell types.Finally,we showed that inflammation activated Jak1–Stat3 signaling in MG,promoting their transition from a resting to an activated state.Our study reveals the cell heterogeneity and crosstalk of immune cells and MG in zebrafish retinal repair,and may provide valuable insights into future mammalian retina regeneration.
基金supported by the Polish National Science Center research grants:OPUS 16[grant number:2018/31/B/NZ9/00781]OPUS 20[grant number:2020/39/B/NZ9/01061]and SONATA 16[grant number:2020/39/D/NZ9/01009]。
文摘Background The pituitary belongs to the most important endocrine glands involved in regulating reproductive functions.The proper functioning of this gland ensures the undisturbed course of the oestrous cycle and affects the female's reproductive potential.It is believed that visfatin,a hormone belonging to the adipokine family,may regulate reproductive functions in response to the female's metabolic state.Herein we ve rified the hypothesis that suggests a modulatory effect of visfatin on the anterior pituitary transcriptome during the mid-luteal phase of the oestrous cycle.Results RNA-seq analysis of the porcine anterior pituitary cells revealed changes in the expression of 202 genes(95 up-regulated and 107 down-regulated in the presence of visfatin,when compared to the non-treated controls),assigned to 318 gene ontology terms.We revealed changes in the frequency of alternative splicing events(235 cases),as well as long noncoding RNA expression(79 cases)in the presence of the adipokine.The identified genes were associated,among others,with reproductive system development,epithelial cell proliferation,positive regulation of cell development,gland morphogenesis and cell chemotaxis.Conclusions The obtained results indicate a modulatory influence of visfatin on the regulation of the porcine transcriptome and,in consequence,pituitary physiology during the mid-luteal phase of the oestrous cycle.
文摘目的利用二代测序技术筛选出具有显著性变化的基因,并探讨月腺大戟素A在转录组学层面上抗乳腺癌活性的作用机制。方法从月腺大戟药材中提取乙酰间苯三酚类化合物月腺大戟素A,对MCF-7细胞(乳腺癌细胞的luminal A型)进行干扰,观察被干扰后的细胞与正常细胞差异基因表达,采用二代高通量测序平台(IlluminaHi-Seq)测序技术分别对对照组和实验组各3个样本进行高通量转录组测序并进行数据分析。结果对照组和实验组分别总获得123656848、123974262个干净序列(clean reads),分别对比到参考基因组上的序列为119762214、119881622,各占总数的96.85%、96.69%;2组转录组对照可得:差异基因总数为1695个,其中上调基因770个,下调基因925个,可清楚注释的基因有3874个。应用基因本体论(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)进行生物功能富集分析,GO分析发现这3874个基因主要涉及生物过程(1270个)、细胞组成(1322个)与分子功能(1282个)3个大类的45个小类,包括细胞的生长发育过程、信号蛋白活性、膜以及基因表达的调控等过程;KEGG分析发现差异表达基因涉及263条信号通路,主要代谢通路为PI3K-Akt信号通路、MAPK信号通路;以及碳水化合物代谢、心肌系统和细胞生殖系统等生物过程。结论利用二代高通量测序平台测序技术一共筛选、鉴定出差异基因1695个,更深入了解了月腺大戟素A与MCF-7细胞基因之间的相互关系,为乳腺癌治疗提供了一些理论基础。
基金This work was supported by NIH/NIDCR grants R01DE029173 and R01DE030445 and NIH/NCI grant R01CA236878.
文摘RNA sequencing(RNAseq)can reveal gene fusions,splicing variants,mutations/indels in addition to differential gene expression,thus providing a more complete genetic picture than DNA sequencing.This most widely used technology in genomics tool box has evolved from classic bulk RNA sequencing(RNAseq),popular single cell RNA sequencing(scRNAseq)to newly emerged spatial RNA sequencing(spRNAseq).Bulk RNAseq studies average global gene expression,scRNAseq investigates single cell RNA biology up to 20,000 individual cells simultaneously,while spRNAseq has ability to dissect RNA activities spatially,representing next generation of RNA sequencing.This article highlights these technologies,characteristic features and suitable applications in precision oncology.
基金supported by National Key Research and Development Program of China(2018YFD1000300)the National Natural Science Foundation of China(31772273)Hubei Provincial Natural Science Foundation for Innovative Group(2017CFA018).
文摘Polyploid plants often exhibit enhanced stress tolerance relative to their diploid counterparts,but the physiological and molecular mechanisms of this enhanced stress tolerance remain largely unknown.In this study,we showed that autotetraploid trifoliate orange(Poncirus trifoliata(L.)Raf.)exhibited enhanced salt tolerance in comparison with diploid progenitors.Global transcriptome profiling of diploid and tetraploid plants with or without salt stress by RNAseq revealed that the autotetraploids displayed specific enrichment of differentially expressed genes.Interestingly,the leaves and roots of tetraploids exhibited different expression patterns of a variety of upregulated genes.Genes related to plant hormone signal transduction were enriched in tetraploid leaves,whereas those associated with starch and sucrose metabolism and proline biosynthesis were enriched in roots.In addition,genes encoding different antioxidant enzymes were upregulated in the leaves(POD)and roots(APX)of tetraploids under salt stress.Consistently,the tetraploids accumulated higher levels of soluble sugars and proline but less ROS under salt stress compared to the diploids.Moreover,several genes encoding transcription factors were induced specifically or to higher levels in the tetraploids under salt stress.Collectively,this study demonstrates that the activation of various multifaceted defense systems in leaves and roots contributes to the enhanced salt tolerance of autotetraploids.
基金This work was supported by Italian Ministery of Health RC 08C921 to LL,Istituto Auxologico Italiano,IRCCs.
文摘Taking advantage of the fast-growing knowledge of RNA-binding proteins(RBPs)we review the signature of downregulated genes for RBPs in the transcriptome of induced pluripotent stem cell neurons(iNeurons)modelling the neurodevelopmental Rubinstein Taybi Syndrome(RSTS)caused by mutations in the genes encoding CBP/p300 acetyltransferases.We discuss top and functionally connected downregulated genes sorted to“RNA processing”and“Ribonucleoprotein complex biogenesis”Gene Ontology clusters.The first set of downregulated RBPs includes members of hnRNHP(A1,A2B1,D,G,H2-H1,MAGOHB,PAPBC),core subunits of U small nuclear ribonucleoproteins and Serine-Arginine splicing regulators families,acting in precursor messenger RNA alternative splicing and processing.Consistent with literature findings on reduced transcript levels of serine/arginine repetitive matrix 4(SRRM4)protein,the main regulator of the neural-specific microexons splicing program upon depletion of Ep300 and Crebbp in mouse neurons,RSTS iNeurons show downregulated genes for proteins impacting this network.We link downregulated genes to neurological disorders including the new HNRNPH1-related intellectual disability syndrome with clinical overlap to RSTS.The set of downregulated genes for Ribosome biogenesis includes several components of ribosomal subunits and nucleolar proteins,such NOP58 and fibrillarin that form complexes with snoRNAs with a central role in guiding post-transcriptional modifications needed for rRNA maturation.These nucleolar proteins are“dual”players as fibrillarin is also required for epigenetic regulation of ribosomal genes and conversely NOP58-associated snoRNA levels are under the control of NOP58 interactor BMAL1,a transcriptional regulator of the circadian rhythm.Additional downregulated genes for“dual specificity”RBPs such as RUVBL1 and METTL1 highlight the links between chromatin and the RBP-ome and the contribution of perturbations in their cross-talk to RSTS.We underline the hub position of CBP/p300 in chromatin regulation,the impact of its defect on neurons’post-transcriptional regulation of gene expression and the potential use of epidrugs in therapeutics of RBP-caused neurodevelopmental disorders.
基金the financial support provided by the National Program on Key Research Project of China(2016YFD0500406)the National Natural Sciences Foundation of China(Grant No.31872455)+1 种基金the Fundamental Research Funds for the Central Universities(2662018PY016)the Start-up Research Fund from Huazhong Agricultural University.
文摘Influenza viruses not only cause respiratory illness,but also have been reported to elicit neurological manifestations following acute viral infection.The central nervous system(CNS)has a specific defense mechanism against pathogens structured by cerebral microvasculature lined with brain endothelial cells to form the blood–brain barrier(BBB).To investigate the response of human brain microvascular endothelial cells(hBMECs)to the Influenza A virus(IAV),we inoculated the cells with the A/WSN/33(H1N1)virus.We then conducted an RNAseq experiment to determine the changes in gene expression levels and the activated disease pathways following infection.The analysis revealed an effective activation of the innate immune defense by inducing the pattern recognition receptors(PRRs).Along with the production of proinflammatory cytokines,we detected an upregulation of interferons and interferon-stimulated genes,such as IFN-β/λ,ISG15,CXCL11,CXCL3 and IL-6,etc.Moreover,infected hBMECs exhibited a disruption in the cytoskeletal structure both on the transcriptomic and cytological levels.The RNAseq analysis showed different pathways and candidate genes associated with the neuroactive ligand-receptor interaction,neuroinflammation,and neurodegenerative diseases,together with a predicted activation of the neuroglia.Likewise,some genes linked with the mitochondrial structure and function displayed a significantly altered expression.En masse,this data supports that hBMECs could be infected by the IAV,which induces the innate and inflammatory immune response.The results suggest that the influenza virus infection could potentially induce a subsequent aggravation of neurological disorders.
文摘Background:To treat vascular proliferative diseases,anti-VEGF therapies have shown systemic adverse effects attributable to the lack of selectivity between pathological and physiological angiogenesis.Thus,identifying the molecular mechanisms that are only specific to pathological cell types is crucial to develop better precision medicine.Methods:Here,we used different cell type enrichment approaches combined with single-cell RNA sequencing to define the transcriptomic changes within each retinal cell types in a mouse model of ischemic retinopathy.This retinal model develops pathological neovascularization(NV)in response to local hypoxia following oxygen-induced vessel obliteration(P7 to P12).The NV phenotype is characterized by the progressive appearance of vascular tufts resulting from misguided,abnormal proliferation of endothelial cells that we monitored at 3 consecutive time points-P12,P14 and P17(peak of NV).Results:By following the dynamic response to hypoxia,our experimental design reveals how pathological angiogenesis is specifically associated with significant metabolic adaptations in different subtypes of endothelial cells(i.e.,Tips vs Stalk cells).We also identify a pathological subtype of glial cells over-expressing VEGFA and pro-inflammatory IL-1 receptor subunits.This subtype of activated glial cells was targeted using selective IL1R antagonist treatment which reduced glial activation,inflammation,NV and promotes physiological angiogenesis,therefore improving tissue regeneration.Conclusions:Our results illustrate how analyzing cell type heterogeneity in tissues developing pathological angiogenesis allows establishing better targeting therapies to restore vascular integrity.
文摘目的分析比较不同肿瘤基质评分胃癌患者的基因表达特征,鉴定与评分相关的胃癌预后基因,以期为临床胃癌诊断和预后提供更精准的手段。方法从癌症基因组图谱数据库(the cancer genome atals,TCGA)下载胃癌的临床资料和组织转录组测序(ribonucleic acid sequencing,RNAseq)表达数据。从基质免疫评估数据库(estimation of stromal and immune cells in malignant tumor tissues using expression data,ESTIMATE)网站下载TCGA数据库中胃癌患者基质评分信息。获取患者的临床信息、RNAseq表达谱、基质评分。按照基质评分的高低分为高基质评分组和低基质评分组,分析基质评分与胃癌预后的关系。用R语言DEseq2包进行标准化处理和差异分析;WGCNA(weight correlation network analysis,WGCNA)包筛选与基质评分密切相关的差异基因;单因素COX风险比例回归模型(COX proportional model,COX)初步筛选基质评分密切相关基因中与胃癌预后相关的基因;LASSO(least absolute shrinkage and selection operator,LASSO)回归模型筛选出其中影响胃癌预后的关键基因,计算最小λ值;多因素COX回归分析构建关键基因胃癌预后模型,并量化基因表达量与患者生存时间的关系;模型内部绘制关键基因的生存曲线。最后通过其他公共数据库(KM-plotter数据库和Oncomine数据库)验证这些基因在胃癌大样本的表达和预后。结果基质评分越高的患者表现为预后更差(P<0.05)。对患者的RNA-seq差异表达分析筛选得到1581个差异表达基因;从中通过WGCNA筛选出1015个基因与胃癌基质评分密切相关;单因素COX回归选出377个基因与胃癌患者预后相关(P<0.05);LASSO回归筛选出12个与胃癌预后相关的关键基因,最小λ=12;多因素COX回归分析显示该模型C指数为0.68,3年生存期和5年生存期的预测值基本贴合实际值,3年生存期曲线下面积(area under the curve,AUC)为0.693,5年生存时间AUC为0.725。12个基因中,ACTA1、ADAMTS12、LINCO1614、MATN3、MTUS2、PLCL1、POSTN、SERPINE1、TPTEP1表达量越高,患者生存期越短,GAD1和MMP16表达量的越低,患者生存期越短;6个基因(ADAMTS12、MATN3、MEGF10、PLCL1、POSTN、SERPINE)各自作为独立危险因素,具有最佳的胃癌预后预测功能(P<0.05)。KM-plotter数据库和Oncomine数据库符合本研究的预测结果。结论肿瘤基质评分越高的胃癌患者,预后更差、生存周期更短。6个基因ADAMTS12、MEGF10、PLCL1、POSTN、MATN3、SERPINE与患者的肿瘤基质评分及预后密切相关。其表达越高,患者评分越高,预后越差、生存周期越短。本研究鉴定了与胃癌基质评分相匹配的预后基因,提示胃癌基质研究的进一步方向。
