| Background and aims: The hepatic fibrosis is a dynamic and dysbalanced wound-healing response caused by chronic liver injury,which is characterized by the net accumulation of extracellular matrix(ECM).Cirrhosis is the pathological state of the late stage of liver fibrosis.The global burden of liver cirrhosis is severe,and antifibrosis research has become a hot and difficult topic in the field of liver disease.HSCs are considered to be the main ECM-producing cell population.The transformation of HSCs from a quiescent,vitamin-A storing state into an activated,proliferative state represents continual liver damage and the formation of fibrosis.So it play a central role in the development of liver fibrosis and cirrhosis.Enhancing understanding of HSCs activation mechanisms and fibrogenesis is conducive to the early diagnosis of hepatic fibrosis and the discovery of potential therapeutic targets.The emergence of high throughput sequencing technology has brought unprecedented opportunities for the identification of key molecules based on transcriptomics.In addition to coding RNAs,nc RNAs are also involved in regulating the activation of HSCs.In view of the fact that the exact mechanisms of hepatic fibrosis have not yet been established and no transcriptome studies of patient-derived HSCs based on the NGS technology are available,this study aimed to demonstrate the separation technology of human HSCs and to analyze the expression of m RNAs and mi RNAs in human HSCs by RNA sequencing(RNA-seq)technology,so as to find the molecular targets related to the diagnosis and treatment of hepatic fibrosis.Nowadays,“-omics” studies are looking at the characterization of single cells,leading to potential and unexpected biological discoveries for researchers.The purpose of this study was to investigate the changes of cell subsets and potential regulatory mechanisms in HBV-related cirrhosis and to reveal the disease essence by single-cell sequencing technology.Materials and methods:In this study,human HSCs were isolated from liver tissues after surgical resection by collagenase type Ⅳ’s recirculating perfusion.The spontaneous fluorescence of vitamin A and immunofluorescence staining were used for the identification of HSCs.HSCs from 9 cirrhotic and 6 non-cirrhotic patients were collected to form the cirrhotic-noncirrhotic HSCs comparison scheme(non-cirrhotic HSCs as control),and the differences of m RNA-omics of HSCs between cirrhotic and non-cirrhotic states were compared.Freshly isolated HSCs in good condition were cultured in vitro for 14 days and 7 pairs of HSCs in the initial and fully activated states were collected,which constituted the activated-freshly isolated HSCs comparison scheme,to simulate the process of hepatic fibrosis and compare gene expression of HSCs before and after culture.The specific steps are as follows: The demographic and clinical characteristics of study subjects were collected,and the warm ischemia time and yield of HSCs were recorded.Extraction of RNAs,library preparation of c DNAs and sequencing were performed on HSCs from the above two schemes.The expression levels of the sequencing data were calculated and the correlations of m RNAs between samples and differentially expressed m RNAs(DEm RNAs)were obtained.The expression patterns of DEm RNAs in different samples were determined by hierarchical clustering analysis.The enrichment analyses(GO,KEGG,Reactome and Dis Ge NET)and the data consistency test were carried out on DEm RNAs of the two schemes.The hub genes associated with hepatic fibrosis were searched by protein-protein interaction(PPI)network and verified by the quantitative real time PCR(q RT-PCR).4 pairs of HSCs before and after in vitro culture were selected,and basic information of patients and liver tissues was collected.Extraction of RNAs,library preparation of c DNAs and sequencing were performed on HSCs.The expression levels of mi RNAs and the correlations between samples were calculated.Differential expression analysis and hierarchical clustering analysis were performed on mi RNAs and the data in this study were compared with the publicly available mi RNA-seq data of liver tissues.The q RT-PCR was used to validate mi RNAs associated with liver diseases.The targeted m RNAs of differentially expressed micro RNAs(DEmi RNAs)were predicted to construct the co-expression networks and GO and KEGG enrichment analysis of the targeted DEGs were performed.The role of mi R-1268 a and mi R-665 in hepatic fibrosis was preliminarily analyzed.Single-cell suspension was obtained by recirculating perfusion and sample preparation process of single-cell sequencing.4 HBV-related cirrhotic and 3 normal liver tissues were included to form the single-cell’ Gel Bead in Emulsions(GEMs)structure on the 10× Chromium Controller.Subsequently,library construction,sequencing,quality assessment of single-cell sequencing and generation of cell-gene expression matrices were performed.After cell filtration,correction,dimensionality reduction and clustering for the expression matrices of multiple samples were done.Cell types of different clusters were defined by cell markers.Results:HSCs from human liver tissues were successfully isolated in this study,with cell yield ranging from 105 to 106 cells/g.In mRNA-seq,gender,age distribution and liver function indexes between cirrhotic and non-cirrhotic patients were equally comparable.1 cirrhotic sample was removed due to inconsistent clustering during analysis.Finally,3,828 DEGs were screened from HSCs of cirrhotic tissues,among which 2,251 and 1,577 genes were significantly up-regulated and down-regulated,respectively.The paired samples(7 pairs)were included in activated-freshly isolated HSCs comparison scheme,which could eliminate the interferences of the disease basis or other indicators.The expression of m RNAs changed significantly after activation of HSCs,and a total of 2,262 DEGs were identified,including 1,032 significantly up-regulated genes and 1,230 significantly down-regulated genes.The hierarchical clustering analysis of the two schemes showed that the expression patterns of genes were similar under the same treatment condition,moreover,the classical genes related to hepatic fibrosis could be verified in differential expression profiles.According to the results of bioinformatics analyses of the two schemes,DEGs participated in ECM composition,small molecule metabolism and other processes,and were significantly enriched in many pathways,such as focal adhesion,retinol metabolism and formation,assembly or degradation of collagen or ECM.PPI analysis showed that there were 9,960 and 8,042 pairs of interactions in cirrhotic-noncirrhotic HSCs comparison scheme and activated-freshly isolated HSCs comparison scheme,respectively.17 genes(COL1A1,COL1A2,VIM,MMP2,ITGB4,ITGA2,CDK1,HGF,CAV1,GSTP1,GNAI2,APP,SHC1,LPL,BCR,ESR1 and CXCR4)were identified as hub genes of hepatic fibrosis,including genes with clear relationships with hepatic fibrosis(COL1A1,MMP2,HGF,etc.)and genes with unclear functions.The results of q RT-PCR suggested that above genes were changed at transcription levels when cirrhosis occurred or HSCs were activated in vitro,reflecting the response of HSCs in the progression of hepatic fibrosis.According to mi RNA-seq analysis,a total of 230 DEmi RNAs were determined,from which 118 were up-regulated and 112 were down-regulated upon HSCs activation.From the mi RNAs,which showed the most significant differences in expression,it was found that liver disease-related mi RNAs such as mi R-758-3p,mi R-493-5p,mi R-409-3p,mi R-31-5p,mi R-1268 a and mi R-381-3p might play a role in hepatic fibrosis.Among them,mi R-1268 a,which possessed the richest targeted DEm RNAs,was vital in the regulation of HSCs activation.After comparing the HSCs’ sequencing data in this study with the publicly available liver tissues’ sequencing data,let-7g-5p,mi R-101-3p,mi R-107,mi R-122-5p,mi R-127-3p,mi R-139-5p,mi R-148a-3p,mi R-192-5p,mi R-194-5p,mi R-215-5p,mi R-24-3p,mi R-26a-5p,mi R-340-5p,mi R-451 a and mi R-99a-5p were verified to overlap between two groups.Mi R-101-3p,mi R-192-5p and mi R-24-3p with contradictory expression levels were tested and results were basically consistent with our data.Meanwhile,co-expression networks of mi RNAs-m RNAs were described(|pearson correlation coefficient|≥0.7 and p<0.05)and the network consisted of 1,891 matched mi RNA-m RNA pairs representing 138 DEmi RNAs and 1,414 DEm RNAs(mi R-665 exhibiting the highest connection).The targeted DEGs were involved in collagen metabolism,ECM structural constituent,cytoskeletal protein binding,cell adhesion and other processes.The expression of COL1A1 could be up-regulated by silencing mi R-1268 a or introducing mi R-665 mimic in LX-2 cells.Based on the differences of gene expression in single cells,64,990 cells from 7 human liver tissues were clustered into 14 clusters,each cluster containing cells from cirrhotic and normal liver tissues.The preliminary classification and definition of cells were carried out with the reference databases and 9 cell types were identified,including hepatocytes,T lymphocytes,B lymphocytes,mononuclear macrophages,cholangiocytes,endothelial cells,mesenchymal cells,innate lymphoid cells(ILCs)and dendritic cells(DCs).The clustering heatmap showed that the gene expression in clusters was in a ladder shape with clear differences and the corresponding functions of different cell populations were performed in HBV-related cirrhosis.Conclusions:By recirculating perfusion method established in this study,liver tissues could be fully digested and human HSCs could be successfully isolated,which has laid a foundation for the study on the mechanisms of hepatic fibrosis.Using RNA-seq technology,this study revealed the changing characteristics of human HSCs during disease progression or in vitro activation from the levels of m RNAs and mi RNAs,providing the basis and reference for the selection of diagnostic and therapeutic targets of hepatic fibrosis.The hub genes and mi RNAs mentioned above were closely related to the activation of HSCs or the occurrence of fibrosis and might become candidate biomarkers.The regulatory mechanisms of hepatic fibrosis are complex,which need to be explored by researchers.In addition,this study established a single-cell atlas of HBV-associated cirrhosis based on different cell populations in the liver.Future research will focus on the use of single-cell sequencing results and correlation analysis to dissect unanticipated aspects of the cellular and molecular basis of hepatic fibrosis and provide the conceptual framework required to study therapeutic targets in hepatic fibrosis. |
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