Identification Of Cirrhosis Or Hepatocellular Carcinoma-related Cell Types And Functional Analysis Based On Single-cell Sequencing And Genome-wide Association Analysis Data

Section 1 Identification of cirrhosis anord hepatocellular carcinoma-related cell typesChronic liver disease is a global public health problem.It is estimated that about 844 million people worldwide suffer from chronic liver disease,causing about 2 million deaths each year.Chronic liver disease usually begins with chronic hepatitis caused by various causes.After repeated liver injury,it can gradually develop into liver cirrhosis and even hepatocellular carcinoma(hepatocellular carcinoma,HCC).In recent years,with the development of molecular biology,more and more attention has been paid to the role of host genetic factors in liver cirrhosis and HCC.Genome-wide association study(Genome-wide association studies,GWASs)promotes our understanding of the genetic role of cirrhosis and HCC.While single-cell RNA sequencing(sc RNAseq)reveals the liver cellular composition and functional characteristics under the physiological and pathological conditions with unprecedented resolution.However,at present,the understanding of liver cirrhosis and HCC is far from enough,and further revealing its disease process is still of great significance for exploring new treatment strategies.Objective: Make integrative analysis on the GWAS and sc RNA-seq data of cirrhosis and HCC to uncover disease-related cell types and provide clues for understanding on the pathogenesis.Methods: We downloaded three GWAS summary data on cirrhosis and HCC from Bio Bank Japan and Gene ATLAS,and three sc RNA-seq data from GEO database.We also referred to the Ensembl database(GRCh37)for SNP and gene annotation information.After processing and defining the cell types for each sc RNA-seq data,we used Roly Poly and LDSC-cts to integrate the GWAS and sc RNA-seq.We also changed the resolution used in clustering and the number of feature genes included to make a sensitivity analysis.In addition,we analyzed one sc RNA-seq data without association to liver diseases to valid the specificity of our findings.Results: After processing the sc RNA-seq data,we obtain about 19,002~32,200 cells and identified 10~17 cell types.For the HCC analysis,we identified the association between B cell and HCC in two datasets.Roly Poly also identified the association,when we integrated the two sc RNA-seq datasets.The sensitivity analysis also support these findings.In specificity analysis,we identified no significant cell type associated with HCC.As for the cirrhosis analysis,we obtained no significant related cell type.Conclusion: In this integrative analysis,we identified B cell as HCC-related cell type.More attention and verification should be paid to them in future research.Section 2 Functional analysis of hepatocellular carcinoma-related cell typesB lymphocytes is an important component of the adaptive immune system,which also plays a key role in the regulation of the host’s immune response.Previous studies showed that immune infiltration of B cells in the liver tissue,and the secretion of immunoglobulin and complement deposition,was associated with the occuarence and development of viral hepatitis-related acute liver failure,primary biliary cholangitis,and non-alcoholic fatty hepatitis.The prebious part also suggested that B cell may be a HCC-related cell type.In this part,we focused on the functions of B cells in HCC and normal liver tissues,to explore the potential association between B cells and HCC.Objective: To make analysis on the cell types associated with liver cirrhosis and HCC identified in the first part.Methods: Sc RNA-seq data of HCC and non-tumor control samples were downloaded from GEO database and processed.We extracted B cells and performed differential expression analysis,in which those with |log FC| < 0.25 and adjusted P < 0.05 were regarded as differentially expressed genes.Based on KEGG,GO,Biocarta and Reactome databases,pathway enrichment and gene set variation analysis analysis were performed.Based on the Cell Phone DB ligand-receptor repository,interactions between B cells and other cell types in HCC and non-tumor control samples were analyzed.Results: A total of 1196 B cells were obtained from 10 primary tumor samples and 8 non-tumor samples.The HCC group has a higher proportion of memory B cells and a relatively lower proportion of activated B cells.A total of 105 genes were significantly up-regulated and 40 genes were significantly down-regulated in HCC group by gene differential expression analysis.Functional enrichment analysis and gene set variation analysis showed that biological processes like coagulation and hemostasis and lipid metabolism related pathways were significantly enriched in B cells of the HCC group.The number of ligand-receptor pairs between B cells and other cell types in HCC group was higher than that in non-tumor group.Ligand-receptor relationships such as LTB-LTBR and SPP1-CD44 were significantly enriched between several cell types and B cells in the HCC group.Conclusion: There were significant differences in composition and functional expression between B cells from HCC and non-tumor liver tissue.B cells may interact with other cell types in liver tissue through ligand receptor pathways like SPP1-CD44 and LTBR-LTB,which may be potentially related to the occurrence of HCC.