Transcriptome Analysis And Related Mechanisms Of Liver Cirrhosis

Background and aims:Liver cirrhosis is a major disease threatening human health.The development of liver cirrhosis is a dynamic process involving many molecular and cellular processes.As two main types of liver parenchymal cells,liver sinusoidal endothelial cells(LSECs)and hepatic stellate cells(HSCs)play important roles in liver cirrhosis.LSECs constitute the wall of hepatic sinusoid.As the first to be impacted by injury,LSECs participate in the pathogenesis of various liver diseases caused by different etiologies.Transcriptome analysis is a powerful tool to search for differential gene expression and further explore functions of these genes.Combining RNA sequencing(RNA-seq)and single cell RNA sequencing(sc RNA-seq)to explore changes in gene expression of LSECs during cirrhosis can provide new directions for anti-liver fibrosis therapy targeted on LSECs.By secreting collagen and other related components,HSCs directly participate in the deposition of extracellular matrix(ECM),whose proliferation and activation play crucial roles in liver cirrhosis.The whole transcriptome sequencing analysis of HSCs before and after culture-induced activation was performed in our previous research.We found that miR-654-5p expression is significantly upregulated,while RXR?expression is downregulated in culture-activated human primary HSCs in vitro.As an important nuclear receptor,RXR?plays a key role in regulating the activation and proliferation of HSCs.In addition,a variety of micro RNAs(miRNAs)serve as important regulatory factors of liver fibrosis and cirrhosis,and miR-654-5p is involved in cell migration and proliferation.By exploring the effects of miR-654-5p and RXR?on the activation and proliferation of HSCs and their specific regulatory mechanism,it is beneficial to the anti-fibrosis treatment targeted on HSCs.Materials and methods:In this study,a single cell suspension containing all kinds of hepatic cells was prepared by collagenase circulating perfusion method,and LSECs and HSCs were further isolated by density gradient centrifugation and immunomagnetic bead sorting.The isolated cells were identified by morphology and immunofluorescence staining.Purities of cells were detected by flow cytometry.LSECs came from 7 non-cirrhotic patients and 4 cirrhotic patients were included in RNA-seq,and transcriptional information between the two groups was compared,and the differentially expressed genes(DEGs)during liver cirrhosis were analyzed.In sc RNA-seq,liver tissues from 3 non-cirrhotic patients and 4 cirrhotic patients were included to prepare single cell suspensions containing all types of hepatic cells.After obtaining the sequencing data,LSECs were identified by specific gene expression patterns,and their DEGs between the cirrhotic group and the non-cirrhotic group were also compared.Then,results of RNA-seq and sc RNA-seq were compared to screen the genes with the same expression in the two schemes.Quantitative real time PCR(q RT-PCR),Western Blotting(WB)and tissue immunofluorescence were performed on some genes with obvious differences,so that to verify the differences in transcription and protein expression levels.In terms of the study on HSCs proliferation and activation mechanism,HSCs from cirrhotic or non-cirrhotic patients were isolated and cultured in vitro for 14 days to induce spontaneous activation.At the same time,recombinant human TGF-?1 was used to stimulate LX-2 cells to establish activated cell model in vitro.m RNAs and miRNAs were extracted from quiescent and activated cells,respectively.miR-654-5p,RXR?and activation-related genes in HSCs,col1a1 and MMP2,were compared by q RT-PCR in the two groups.After transfected with miR-654-5p mimics,the expression changes of above genes in LX-2 cells were estimated via q RT-PCR and WB.Cell proliferation and apoptosis were detected in miR-654-5p overexpressed LX-2 cells through CCK-8 assay and flow cytometry.Subsequently,Target Scan database was used to predict possible the targeting interaction and binding sites between miR-654-5p and RXR?.In order to further verify the direct targeting relationship between the two,wild type RXR?3'UTR and its mutant sequence were constructed into reporter plasmid to complete the dual luciferase reporter assay.Then,LX-2 cells were transfected with RXR?overexpression plasmid or its control,miR-654-5p mimics were co-transfected at the same time.In order to estimate whether the overexpression of RXR?rescued the effect miR-654-5p mimics on HSCs,the expression levels of activation-related genes,cell proliferation and apoptosis in HSCs were compared between the two groups.In addition,liver fibrosis in C57BL/6J mice was induced by intraperitoneal injection of carbon tetrachloride(CCl₄)for 6 weeks.q RT-PCR were used to compare the expression levels of miR-654-5p,RXR?and activation-related genes between vehicle and CCl₄ group.In order to study the specific role of miR-654-5p during liver cirrhosis in vivo,adeno-associated viruses packed with miR-654-5p gene or its control were injected into mice through the tail vein before the six-week abdominal injection of CCl₄.After the molding is finished,mouse liver tissues were obtained,and the degree of fibrosis between the two groups was compared by Masson staining and hydroxyproline determination.Results:In this study,high purity human primary LSECs and HSCs were successfully isolated from the patient's surgically resected liver tissues.In transcriptome analysis of LSECs in 7 patients without liver cirrhosis and 4patients with cirrhosis,a total of 1987 DEGs were obtained by RNA-seq,and 461 DEGs were obtained by sc RNA-seq.Further screening was performed after comparison of these two parts of DEGs.After comparison between the two parts of DEGs,further screening was carried out through changes in expression level,and a 24 DEGs with obvious differences were finally retained.There were 19 up-regulated genes(FGF23,CH25,ANGPTL4,IL1RL1,CYP3A7,SERPINE1,F2RL3,MT1M,APOLD1,ADM,MFAP4,ERRFI1,CD9,LCN6,AKAP12,PLIN2,BNIP3,CAV2,MT1A)and 5 down-regulated genes(GPR182,HSPA1B,RGS16,HSPA6,MT-ND4L).Some of the genes related to liver disease or vascular disease were selected for q RT-PCR experiment,which further verified the accuracy of the sequencing results.Among them,FGF23m RNA expression level was the most up-regulated during cirrhosis.Correspondingly,its protein level in LSECs from cirrhotic liver was also increased.In the study of roles of miR-654-5p and RXR?in HSCs regulation,we found that miR-654-5p expression level was increased in both activated human HSCs and TGF-?1-treated LX-2 cells,companied with the downregulation of RXR?.In addition,miR-654-5p mimics significantly promoted the expression of activation-related genes in LX-2 cells,as well as promoted cell proliferation and inhibited apoptosis.Accordingly,the overexpression of miR-654-5p in LX-2 cells inhibited the expression of RXR?.The predicted results in Target Scan database showed that there were targeted binding sites between miR-654-5p and RXR?.These predictions were further confirmed by dual luciferase reporter assay.Upregulation of RXR?rescued the effect of miR-654-5p on LX-2 cells.Be consistent with this,in vivo experiments also confirmed the miR-654-5p increase and RXR?decline in CCl₄-induced liver fibrosis.Overexpression of miR-654-5p in vivo aggravates liver fibrosis.Conclusion:Through transcriptome analysis of LSECs,DEGs related to the pathophysiological processes of LSECs during liver cirrhosis were found,which provides possible targets for further researches and anti-fibrotic treatment.By studying the mechanisms of HSCs activation,proliferation and apoptosis,we found that miR-654-5p contribute to HSCs regulation by targeting RXR?both in human and mice.This study provides a promising prospect for the treatment of liver fibrosis through targeting HSCs.