Stem cells(SCs)with their self-renewal and pluripotent differentiation potential,show great promise for therapeutic applications to some refractory diseases such as stroke,Parkinsonism,myocardial infarction,and diabet...Stem cells(SCs)with their self-renewal and pluripotent differentiation potential,show great promise for therapeutic applications to some refractory diseases such as stroke,Parkinsonism,myocardial infarction,and diabetes.Furthermore,as seed cells in tissue engineering,SCs have been applied widely to tissue and organ regeneration.However,previous studies have shown that SCs are heterogeneous and consist of many cell subpopulations.Owing to this heterogeneity of cell states,gene expression is highly diverse between cells even within a single tissue,making precise identification and analysis of biological properties difficult,which hinders their further research and applications.Therefore,a defined understanding of the heterogeneity is a key to research of SCs.Traditional ensemble-based sequencing approaches,such as microarrays,reflect an average of expression levels across a large population,which overlook unique biological behaviors of individual cells,conceal cell-to-cell variations,and cannot understand the heterogeneity of SCs radically.The development of high throughput single cell RNA sequencing(scRNA-seq)has provided a new research tool in biology,ranging from identification of novel cell types and exploration of cell markers to the analysis of gene expression and predicating developmental trajectories.scRNA-seq has profoundly changed our understanding of a series of biological phenomena.Currently,it has been used in research of SCs in many fields,particularly for the research of heterogeneity and cell subpopulations in early embryonic development.In this review,we focus on the scRNA-seq technique and its applications to research of SCs.展开更多
The corneal epithelium is composed of stratified squamous epithelial cells on the outer surface of the eye,which acts as a protective barrier and is critical for clear and stable vision.Its continuous renewal or wound...The corneal epithelium is composed of stratified squamous epithelial cells on the outer surface of the eye,which acts as a protective barrier and is critical for clear and stable vision.Its continuous renewal or wound healing depends on the proliferation and differentiation of limbal stem cells(LSCs),a cell population that resides at the limbus in a highly regulated niche.Dysfunction of LSCs or their niche can cause limbal stem cell deficiency,a disease that is manifested by failed epithelial wound healing or even blindness.Nevertheless,compared to stem cells in other tissues,little is known about the LSCs and their niche.With the advent of single-cell RNA sequencing,our understanding of LSC characteristics and their microenvironment has grown considerably.In this review,we summarized the current findings from single-cell studies in the field of cornea research and focused on important advancements driven by this technology,including the heterogeneity of the LSC population,novel LSC markers and regulation of the LSC niche,which will provide a reference for clinical issues such as corneal epithelial wound healing,ocular surface reconstruction and interventions for related diseases.展开更多
BACKGROUND Accumulating evidence suggests that the maxillary process,to which cranial crest cells migrate,is essential to tooth development.Emerging studies indicate that Cd271 plays an essential role in odontogenesis...BACKGROUND Accumulating evidence suggests that the maxillary process,to which cranial crest cells migrate,is essential to tooth development.Emerging studies indicate that Cd271 plays an essential role in odontogenesis.However,the underlying mechanisms have yet to be elucidated.AIM To establish the functionally heterogeneous population in the maxillary process,elucidate the effects of Cd271 deficiency on gene expression differences.METHODS p75NTR knockout(Cd271-/-)mice(from American Jackson laboratory)were used to collect the maxillofacial process tissue of p75NTR knockout mice,and the wildtype maxillofacial process of the same pregnant mouse wild was used as control.After single cell suspension,the cDNA was prepared by loading the single cell suspension into the 10x Genomics Chromium system to be sequenced by NovaSeq6000 sequencing system.Finally,the sequencing data in Fastq format were obtained.The FastQC software is used to evaluate the quality of data and CellRanger analyzed the data.The gene expression matrix is read by R software,and Seurat is used to control and standardize the data,reduce the dimension and cluster.We search for marker genes for subgroup annotation by consulting literature and database;explore the effect of p75NTR knockout on mesenchymal stem cells(MSCs)gene expression and cell proportion by cell subgrouping,differential gene analysis,enrichment analysis and protein-protein interaction network analysis;understand the interaction between MSCs cells and the differentiation trajectory and gene change characteristics of p75NTR knockout MSCs by cell communication analysis and pseudo-time analysis.Last we verified the findings single cell sequencing in vitro.RESULTS We identified 21 cell clusters,and we re-clustered these into three subclusters.Importantly,we revealed the cell–cell communication networks between clusters.We clarified that Cd271 was significantly associated with the regulation of mineralization.CONCLUSION This study provides comprehensive mechanistic insights into the maxillary-process-derived MSCs and demonstrates that Cd271 is significantly associated with the odontogenesis in mesenchymal populations.展开更多
RNA-seq and single-cell genomic research emerge as an important research area in the recent years due to its ability to examine genetic information of any number of single cells in all living organisms.The knowledge g...RNA-seq and single-cell genomic research emerge as an important research area in the recent years due to its ability to examine genetic information of any number of single cells in all living organisms.The knowledge gained from RNA-seq and single-cell genomic research will have a great impact in many aspects of plant biology.In this review,we summary and discuss the biological significance of RNA-seq and single-cell genomic research in plants including the single-cell DNA-sequencing,RNA-seq and single-cell RNA sequencing in woody plants,methods of RNA-seq and single-cell RNA-sequencing,single-cell RNA-sequencing for studying plant development,and single-cell RNA-sequencing for elucidating cell type composition.We will focus on RNA-seq and single-cell RNA sequencing in woody plants,understanding of plant development through single-cell RNAsequencing,and elucidation of cell type composition via single-cell RNA-sequencing.Information presented in this review will be helpful to increase our understanding of plant genomic research in a way with the power of plant single-cell RNA-sequencing analysis.展开更多
Human T cells are a highly heterogeneous population and can recognize a wide variety of antigens by their T cell receptors(TCRs). Tumor cells display a large repertoire of antigens that serve as potential targets for ...Human T cells are a highly heterogeneous population and can recognize a wide variety of antigens by their T cell receptors(TCRs). Tumor cells display a large repertoire of antigens that serve as potential targets for recognition,thus making T cells in the tumor micro-environment more complicated. Making a connection between TCRs and the transcriptional information of individual T cells will be interesting for investigating clonal expansion within T cell populations under pathologic conditions. Advances in single cell RNA-sequencing(scRNA-seq) have allowed for comprehensive analysis of T cells. In this review, we briefly describe the research progress on tumor microenvironment T cells using single cell RNA sequencing, and then discuss how scRNA-seq can be used to resolve immune system heterogeneity in health and disease. Finally, we point out future directions in this field and potential for immunotherapy.展开更多
Background: Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has ...Background: Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has been considered an emerging and potentially toxic pollutant in recent years. Long-term exposure to TCS has been suggested to exert endocrine disruption effects, and promote liver fibrogenesis and tumorigenesis. This study was aimed at clarifying the underlying cellular and molecular mechanisms of hepatotoxicity effect of TCS at the initiation stage.Methods: C57BL/6 mice were exposed to different dosages of TCS for 2 weeks and the organ toxicity was evaluated by various measurements including complete blood count, histological analysis and TCS quantification. Single cell RNA sequencing(scRNA-seq) was then carried out on TCS-or mock-treated mice livers to delineate the TCS-induced hepatotoxicity. The acquired single-cell transcriptomic data were analyzed from different aspects including differential gene expression, transcription factor(TF) regulatory network, pseudotime trajectory, and cellular communication, to systematically dissect the cellular and molecular events after TCS exposure. To verify the TCS-induced liver fibrosis,the expression levels of key fibrogenic proteins were examined by Western blotting, immunofluorescence, Masson’s trichrome and Sirius red stainings. In addition, normal hepatocyte cell MIHA and hepatic stellate cell LX-2 were used as in vitro cell models to experimentally validate the effects of TCS by immunological, proteomic and metabolomic technologies.Results: We established a relatively short term TCS exposure murine model and found the TCS mainly accumulated in the liver. The scRNA-seq performed on the livers of the TCS-treated and control groups profiled the gene expressions of > 76,000 cells belonging to 13 major cell types. Among these types, hepatocytes and hepatic stellate cells(HSCs)were significantly increased in TCS-treated group. We found that TCS promoted fibrosis-associated proliferation of hepatocytes, in which Gata2 and Mef2c are the key driving TFs. Our data also suggested that TCS induced the proliferation and activation of HSCs, which was experimentally verified in both liver tissue and cell model. In addition,other changes including the dysfunction and capillarization of endothelial cells, an increase of fibrotic characteristics in B plasma cells, and M2 phenotype-skewing of macrophage cells, were also deduced from the scRNA-seq analysis, and these changes are likely to contribute to the progression of liver fibrosis. Lastly, the key differential ligand-receptor pairs involved in cellular communications were identified and we confirmed the role of GAS6_AXL interactionmediated cellular communication in promoting liver fibrosis.Conclusions: TCS modulates the cellular activities and fates of several specific cell types(including hepatocytes, HSCs,endothelial cells, B cells, Kupffer cells and liver capsular macrophages) in the liver, and regulates the ligand-receptor interactions between these cells, thereby promoting the proliferation and activation of HSCs, leading to liver fibrosis.Overall, we provide the first comprehensive single-cell atlas of mice livers in response to TCS and delineate the key cellular and molecular processes involved in TCS-induced hepatotoxicity and fibrosis.展开更多
The differential expression of genes in HepG2 cells caused by UC001 kfo RNAi was investigated using RNA-seq. HepG2 cells were infected by Lenti-sh UC001 kfo lentivirus particles. The expression of UC001 kfo m RNA in t...The differential expression of genes in HepG2 cells caused by UC001 kfo RNAi was investigated using RNA-seq. HepG2 cells were infected by Lenti-sh UC001 kfo lentivirus particles. The expression of UC001 kfo m RNA in the HepG2-sh UC001 kfo cell line was detected by real-time PCR. RNA-seq technology was used to identify the difference in the expression of genes regulated by lnc RNA UC001 kfo in the HepG2 cell line. Gene ontology and signaling pathway analysis were performed to reveal the biological functions of the genes encoding of significantly different m RNAs. The results showed that m RNAs were differentially expressed between the HepG2-sh UC001 kfo cell line and the HepG2 cell line. The UC001 kfo m RNA was significantly down-regulated in the stable cell line HepG2-sh UC001kfo（P〈0.001）. Additionally, we found 19 signaling pathways or functional classifications encompassing 30 genes that played a role in cancer characteristics, cell adhesion, invasion and migration. The results also showed that the expression of many genes associated with cancer cell invasion and metastasis was decreased with the down-regulation of the lnc RNA UC001 kfo. Lnc RNA UC001 kfo may play a role in regulating cancer cell invasion and metastasis. It was suggested that m RNAs were differentially expressed in the HepG2 cell line after the down-regulation of lnc RNA-UC001 kfo. Some took part in the extracellular matrix, cell adhesion, motility, growth, and localization. The genes encoding of differentially expressed m RNAs may participate in cell invasion and metastasis.展开更多
The emergence of single-cell RNA-sequencing(scRNA-seq)technology has introduced new information about the structure of cells,diseases,and their associated biological factors.One of the main uses of scRNA-seq is identi...The emergence of single-cell RNA-sequencing(scRNA-seq)technology has introduced new information about the structure of cells,diseases,and their associated biological factors.One of the main uses of scRNA-seq is identifying cell populations,which sometimes leads to the detection of rare cell populations.However,the new method is still in its infancy and with its advantages comes computational challenges that are just beginning to address.An important tool in the analysis is dimensionality reduction,which transforms high dimensional data into a meaningful reduced subspace.The technique allows noise removal,visualization and compression of high-dimensional data.This paper presents a new dimensionality reduction approach where,during an unsupervised multistage process,a feature set including high valuable markers is created which can facilitate the isolation of cell populations.Our proposed method,called fusion of the Spearman and Pearson affinity matrices(FSPAM),is based on a graph-based Gaussian kernel.Use of the graph theory can be effective to overcome the challenge of the nonlinear relations between cellular markers in scRNA-seq data.Furthermore,with a proper fusion of the Pearson and Spearman correlation coefficient criteria,it extracts a set of the most important features in a new space.In fact,the FSPAM aggregates the various aspects of cell-to-cell similarity derived from the Pearson and Spearman metrics,and reveals new aspects of cell-to-cell similarity,which can be used to extract new features.The results of the identification of cell populations via k-means++clustering method based on the features extracted from the FSPAM and different datasets of scRNA-seq suggested that the proposed method,regardless of the characteristics that govern each dataset,enjoys greater accuracy and better quality compared to previous methods.展开更多
Changes in vascular stiffness are associated with the development and progression of many diseases, especially in cardiovascular disease. However, the effect of vascular stiffness on the endothelial cells (ECs) is not...Changes in vascular stiffness are associated with the development and progression of many diseases, especially in cardiovascular disease. However, the effect of vascular stiffness on the endothelial cells (ECs) is not fully understood. Therefore, this study aims to determine the gene expression changes of ECs cultured on the matrices with different stiffness (1 kPa and 40 kPa, respectively) by RNA-seq, thereby broadening the knowledge between mechanics and biology. We obtained 1775 differentially expressed genes (DEGs) by RNA-seq, with 450 up-regulated and 1325 down-regulated DEGs in ECs cultured on soft matrix (1 kPa) compared to those cultured on stiff matrix (40 kPa). After that, we performed a series of functional enrichment analyses based on DEGs and found that DEGs were enriched in many signaling pathways like adhesion junction. Furthermore, transcription factor (TF) target gene prediction analysis and protein-protein interaction (PPI) analysis were also conducted. We found that mechanotransduction signaling related TFs such as BRD4 are involved in. And in the PPI analysis, some genes encoding extracellular matrix proteins such as fibronectin 1 (FN1) were identified as the hub genes. In order to confirm the RNA-seq results, we performed real-time qPCR analysis on the genes of interest, including FN1, collagen α2 (IV) chain, matrix metalloproteinase-14 and integrin α5, and found that the expression levels of all these genes were down-regulated on soft matrix, suggesting that soft matrix caused by pathological conditions may directly attenuate vascular barrier function. This study offers the insights about the effects of physical stimulation on cells, paving a way for vascular tissue engineering, regenerative medicine, disease modeling and therapies.展开更多
基金Supported by the National Natural Science Foundation of China,No.81670951
文摘Stem cells(SCs)with their self-renewal and pluripotent differentiation potential,show great promise for therapeutic applications to some refractory diseases such as stroke,Parkinsonism,myocardial infarction,and diabetes.Furthermore,as seed cells in tissue engineering,SCs have been applied widely to tissue and organ regeneration.However,previous studies have shown that SCs are heterogeneous and consist of many cell subpopulations.Owing to this heterogeneity of cell states,gene expression is highly diverse between cells even within a single tissue,making precise identification and analysis of biological properties difficult,which hinders their further research and applications.Therefore,a defined understanding of the heterogeneity is a key to research of SCs.Traditional ensemble-based sequencing approaches,such as microarrays,reflect an average of expression levels across a large population,which overlook unique biological behaviors of individual cells,conceal cell-to-cell variations,and cannot understand the heterogeneity of SCs radically.The development of high throughput single cell RNA sequencing(scRNA-seq)has provided a new research tool in biology,ranging from identification of novel cell types and exploration of cell markers to the analysis of gene expression and predicating developmental trajectories.scRNA-seq has profoundly changed our understanding of a series of biological phenomena.Currently,it has been used in research of SCs in many fields,particularly for the research of heterogeneity and cell subpopulations in early embryonic development.In this review,we focus on the scRNA-seq technique and its applications to research of SCs.
文摘The corneal epithelium is composed of stratified squamous epithelial cells on the outer surface of the eye,which acts as a protective barrier and is critical for clear and stable vision.Its continuous renewal or wound healing depends on the proliferation and differentiation of limbal stem cells(LSCs),a cell population that resides at the limbus in a highly regulated niche.Dysfunction of LSCs or their niche can cause limbal stem cell deficiency,a disease that is manifested by failed epithelial wound healing or even blindness.Nevertheless,compared to stem cells in other tissues,little is known about the LSCs and their niche.With the advent of single-cell RNA sequencing,our understanding of LSC characteristics and their microenvironment has grown considerably.In this review,we summarized the current findings from single-cell studies in the field of cornea research and focused on important advancements driven by this technology,including the heterogeneity of the LSC population,novel LSC markers and regulation of the LSC niche,which will provide a reference for clinical issues such as corneal epithelial wound healing,ocular surface reconstruction and interventions for related diseases.
基金National Natural Science Foundation of China(General Program),No.31870971Medical Health Science and Technology Project of Zhejiang Province,No.2023KY155.
文摘BACKGROUND Accumulating evidence suggests that the maxillary process,to which cranial crest cells migrate,is essential to tooth development.Emerging studies indicate that Cd271 plays an essential role in odontogenesis.However,the underlying mechanisms have yet to be elucidated.AIM To establish the functionally heterogeneous population in the maxillary process,elucidate the effects of Cd271 deficiency on gene expression differences.METHODS p75NTR knockout(Cd271-/-)mice(from American Jackson laboratory)were used to collect the maxillofacial process tissue of p75NTR knockout mice,and the wildtype maxillofacial process of the same pregnant mouse wild was used as control.After single cell suspension,the cDNA was prepared by loading the single cell suspension into the 10x Genomics Chromium system to be sequenced by NovaSeq6000 sequencing system.Finally,the sequencing data in Fastq format were obtained.The FastQC software is used to evaluate the quality of data and CellRanger analyzed the data.The gene expression matrix is read by R software,and Seurat is used to control and standardize the data,reduce the dimension and cluster.We search for marker genes for subgroup annotation by consulting literature and database;explore the effect of p75NTR knockout on mesenchymal stem cells(MSCs)gene expression and cell proportion by cell subgrouping,differential gene analysis,enrichment analysis and protein-protein interaction network analysis;understand the interaction between MSCs cells and the differentiation trajectory and gene change characteristics of p75NTR knockout MSCs by cell communication analysis and pseudo-time analysis.Last we verified the findings single cell sequencing in vitro.RESULTS We identified 21 cell clusters,and we re-clustered these into three subclusters.Importantly,we revealed the cell–cell communication networks between clusters.We clarified that Cd271 was significantly associated with the regulation of mineralization.CONCLUSION This study provides comprehensive mechanistic insights into the maxillary-process-derived MSCs and demonstrates that Cd271 is significantly associated with the odontogenesis in mesenchymal populations.
文摘RNA-seq and single-cell genomic research emerge as an important research area in the recent years due to its ability to examine genetic information of any number of single cells in all living organisms.The knowledge gained from RNA-seq and single-cell genomic research will have a great impact in many aspects of plant biology.In this review,we summary and discuss the biological significance of RNA-seq and single-cell genomic research in plants including the single-cell DNA-sequencing,RNA-seq and single-cell RNA sequencing in woody plants,methods of RNA-seq and single-cell RNA-sequencing,single-cell RNA-sequencing for studying plant development,and single-cell RNA-sequencing for elucidating cell type composition.We will focus on RNA-seq and single-cell RNA sequencing in woody plants,understanding of plant development through single-cell RNAsequencing,and elucidation of cell type composition via single-cell RNA-sequencing.Information presented in this review will be helpful to increase our understanding of plant genomic research in a way with the power of plant single-cell RNA-sequencing analysis.
基金supported by grants from the National Natural Science Foundation of China (No. 81770152, 91642111 and 81570143)the Guangzhou Science and Technology Project (No. 201510010211, 201807010004 and 201803040017)
文摘Human T cells are a highly heterogeneous population and can recognize a wide variety of antigens by their T cell receptors(TCRs). Tumor cells display a large repertoire of antigens that serve as potential targets for recognition,thus making T cells in the tumor micro-environment more complicated. Making a connection between TCRs and the transcriptional information of individual T cells will be interesting for investigating clonal expansion within T cell populations under pathologic conditions. Advances in single cell RNA-sequencing(scRNA-seq) have allowed for comprehensive analysis of T cells. In this review, we briefly describe the research progress on tumor microenvironment T cells using single cell RNA sequencing, and then discuss how scRNA-seq can be used to resolve immune system heterogeneity in health and disease. Finally, we point out future directions in this field and potential for immunotherapy.
基金supported by the National Key Research and Development Program of China(2020YFA0908000 and 2022YFC2303600)the Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine(ZYYCXTD-C-202002)+12 种基金the National Natural Science Foundation of China(82141001,82274182,82173914,82074098,81903588 and 82003814)the Science and Technology Foundation of Shenzhen(JCYJ20210324115800001)the Science and Technology Foundation of Shenzhen(Shenzhen Clinical Medical Research Center for Geriatric Diseases)the Fundamental Research Funds for the Central Public Welfare Research Institutes(ZXKT18003)the Fundamental Research Funds for the Central public welfare research institutes(ZZ14-YQ-050)the National Key R&D Program of China Key projects for international cooperation on science,technology and innovation(2020YFE0205100)the Shenzhen Governmental Sustainable Development Fund(KCXFZ20201221173612034)the Shenzhen Governmental Sustainable Development Fund(KCXFZ20201221173612034)the Shenzhen key Laboratory of Kidney Diseases(ZDSYS201504301616234)the Shenzhen Fund for Guangdong Provincial High-level Clinical Key Specialties(SZGSP001)the Shenzhen Key Laboratory of Kidney Diseases(ZDSYS201504301616234)the Shenzhen Fund for Guangdong Provincial High-level Clinical Key Specialties(SZGSP001)partially supported by a Grant from the Sanming Project of Medicine in Shenzhen(SZSM201612034).
文摘Background: Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has been considered an emerging and potentially toxic pollutant in recent years. Long-term exposure to TCS has been suggested to exert endocrine disruption effects, and promote liver fibrogenesis and tumorigenesis. This study was aimed at clarifying the underlying cellular and molecular mechanisms of hepatotoxicity effect of TCS at the initiation stage.Methods: C57BL/6 mice were exposed to different dosages of TCS for 2 weeks and the organ toxicity was evaluated by various measurements including complete blood count, histological analysis and TCS quantification. Single cell RNA sequencing(scRNA-seq) was then carried out on TCS-or mock-treated mice livers to delineate the TCS-induced hepatotoxicity. The acquired single-cell transcriptomic data were analyzed from different aspects including differential gene expression, transcription factor(TF) regulatory network, pseudotime trajectory, and cellular communication, to systematically dissect the cellular and molecular events after TCS exposure. To verify the TCS-induced liver fibrosis,the expression levels of key fibrogenic proteins were examined by Western blotting, immunofluorescence, Masson’s trichrome and Sirius red stainings. In addition, normal hepatocyte cell MIHA and hepatic stellate cell LX-2 were used as in vitro cell models to experimentally validate the effects of TCS by immunological, proteomic and metabolomic technologies.Results: We established a relatively short term TCS exposure murine model and found the TCS mainly accumulated in the liver. The scRNA-seq performed on the livers of the TCS-treated and control groups profiled the gene expressions of > 76,000 cells belonging to 13 major cell types. Among these types, hepatocytes and hepatic stellate cells(HSCs)were significantly increased in TCS-treated group. We found that TCS promoted fibrosis-associated proliferation of hepatocytes, in which Gata2 and Mef2c are the key driving TFs. Our data also suggested that TCS induced the proliferation and activation of HSCs, which was experimentally verified in both liver tissue and cell model. In addition,other changes including the dysfunction and capillarization of endothelial cells, an increase of fibrotic characteristics in B plasma cells, and M2 phenotype-skewing of macrophage cells, were also deduced from the scRNA-seq analysis, and these changes are likely to contribute to the progression of liver fibrosis. Lastly, the key differential ligand-receptor pairs involved in cellular communications were identified and we confirmed the role of GAS6_AXL interactionmediated cellular communication in promoting liver fibrosis.Conclusions: TCS modulates the cellular activities and fates of several specific cell types(including hepatocytes, HSCs,endothelial cells, B cells, Kupffer cells and liver capsular macrophages) in the liver, and regulates the ligand-receptor interactions between these cells, thereby promoting the proliferation and activation of HSCs, leading to liver fibrosis.Overall, we provide the first comprehensive single-cell atlas of mice livers in response to TCS and delineate the key cellular and molecular processes involved in TCS-induced hepatotoxicity and fibrosis.
基金supported by National Natural Science Foudation of China(No.U1404309)
文摘The differential expression of genes in HepG2 cells caused by UC001 kfo RNAi was investigated using RNA-seq. HepG2 cells were infected by Lenti-sh UC001 kfo lentivirus particles. The expression of UC001 kfo m RNA in the HepG2-sh UC001 kfo cell line was detected by real-time PCR. RNA-seq technology was used to identify the difference in the expression of genes regulated by lnc RNA UC001 kfo in the HepG2 cell line. Gene ontology and signaling pathway analysis were performed to reveal the biological functions of the genes encoding of significantly different m RNAs. The results showed that m RNAs were differentially expressed between the HepG2-sh UC001 kfo cell line and the HepG2 cell line. The UC001 kfo m RNA was significantly down-regulated in the stable cell line HepG2-sh UC001kfo（P〈0.001）. Additionally, we found 19 signaling pathways or functional classifications encompassing 30 genes that played a role in cancer characteristics, cell adhesion, invasion and migration. The results also showed that the expression of many genes associated with cancer cell invasion and metastasis was decreased with the down-regulation of the lnc RNA UC001 kfo. Lnc RNA UC001 kfo may play a role in regulating cancer cell invasion and metastasis. It was suggested that m RNAs were differentially expressed in the HepG2 cell line after the down-regulation of lnc RNA-UC001 kfo. Some took part in the extracellular matrix, cell adhesion, motility, growth, and localization. The genes encoding of differentially expressed m RNAs may participate in cell invasion and metastasis.
文摘The emergence of single-cell RNA-sequencing(scRNA-seq)technology has introduced new information about the structure of cells,diseases,and their associated biological factors.One of the main uses of scRNA-seq is identifying cell populations,which sometimes leads to the detection of rare cell populations.However,the new method is still in its infancy and with its advantages comes computational challenges that are just beginning to address.An important tool in the analysis is dimensionality reduction,which transforms high dimensional data into a meaningful reduced subspace.The technique allows noise removal,visualization and compression of high-dimensional data.This paper presents a new dimensionality reduction approach where,during an unsupervised multistage process,a feature set including high valuable markers is created which can facilitate the isolation of cell populations.Our proposed method,called fusion of the Spearman and Pearson affinity matrices(FSPAM),is based on a graph-based Gaussian kernel.Use of the graph theory can be effective to overcome the challenge of the nonlinear relations between cellular markers in scRNA-seq data.Furthermore,with a proper fusion of the Pearson and Spearman correlation coefficient criteria,it extracts a set of the most important features in a new space.In fact,the FSPAM aggregates the various aspects of cell-to-cell similarity derived from the Pearson and Spearman metrics,and reveals new aspects of cell-to-cell similarity,which can be used to extract new features.The results of the identification of cell populations via k-means++clustering method based on the features extracted from the FSPAM and different datasets of scRNA-seq suggested that the proposed method,regardless of the characteristics that govern each dataset,enjoys greater accuracy and better quality compared to previous methods.
文摘Changes in vascular stiffness are associated with the development and progression of many diseases, especially in cardiovascular disease. However, the effect of vascular stiffness on the endothelial cells (ECs) is not fully understood. Therefore, this study aims to determine the gene expression changes of ECs cultured on the matrices with different stiffness (1 kPa and 40 kPa, respectively) by RNA-seq, thereby broadening the knowledge between mechanics and biology. We obtained 1775 differentially expressed genes (DEGs) by RNA-seq, with 450 up-regulated and 1325 down-regulated DEGs in ECs cultured on soft matrix (1 kPa) compared to those cultured on stiff matrix (40 kPa). After that, we performed a series of functional enrichment analyses based on DEGs and found that DEGs were enriched in many signaling pathways like adhesion junction. Furthermore, transcription factor (TF) target gene prediction analysis and protein-protein interaction (PPI) analysis were also conducted. We found that mechanotransduction signaling related TFs such as BRD4 are involved in. And in the PPI analysis, some genes encoding extracellular matrix proteins such as fibronectin 1 (FN1) were identified as the hub genes. In order to confirm the RNA-seq results, we performed real-time qPCR analysis on the genes of interest, including FN1, collagen α2 (IV) chain, matrix metalloproteinase-14 and integrin α5, and found that the expression levels of all these genes were down-regulated on soft matrix, suggesting that soft matrix caused by pathological conditions may directly attenuate vascular barrier function. This study offers the insights about the effects of physical stimulation on cells, paving a way for vascular tissue engineering, regenerative medicine, disease modeling and therapies.
