Analysis On Hepatocarcinoma Microenvironment Heterogeneity And Susceptibility Factors Of Stomach For SARS-CoV-2 Based On Single Cell Transcriptome Data

Cellular heterogeneity is a common feature of multicellular organisms.It under normal conditions helps cells achieve specific physiological functions.Starting from the zygote,the gene expression of the cell is regulated by various factors and cells begin to differentiate in different directions,eventually forming cell types that perform specific functions,such as red blood cells responsible for transporting oxygen,muscle cells responsible for exercise,and T cells and B cells for adaptive immune system.The genomes of different types of cells are basically the same,but they have different gene expression patterns and form different tissues,so that they can perform specific functions more professionally.With the development of high-throughput sequencing technology,the method of measuring gene expression at the single cell level has evolved from the original traditional techniques such as q PCR,in situ hybridization and immunohistochemical staining to single-cell RNA sequencing(sc RNA-seq).sc RNA-seq is a sequencing method that can unbiasedly examine the m RNA sequence information and reveal all gene expression information from individual cells.The rapid development of sc RNA-seq in the past decade,especially the large-scale commercialization of the 10X Genomics platform,has further reduced the entry barriers of using sc RNA-seq.Combining new integrated techniques and new ideas enable us to reuse these valuable public resources,to identify and measure the heterogeneity of tissues,to acquire new biological knowledge and to provide important evidence and clues for the diagnosis and treatment of related diseases.In this thesis,the author performed the following research based on integration of the public sc RNA-seq datasets.The first part is analysis on heterogeneity of hepatocarcinoma microenvironment.Hepatocarcinoma,consisting mainly of hepatocellular carcinoma and intrahepatic cholangiocarcinoma,is the second most common cause of cancer death worldwide.The therapeutic failure and high mortality of liver cancer have been proved to be highly related to the heterogeneity of the intricate tumor microenvironment(TME).Thus,resolving the comprehensive cell atlas and molecular component of liver cancer is critical to understanding the liver cancer biology and developing new therapeutic strategies.Here,we performed an integrative analysis and systematically compared the transcriptome within major cell types and cell components between 19 cases of primary liver cancer and5 normal liver samples.We identified two subpopulations of fibroblasts that exclusively expanded in liver cancers and then we described their transcriptomic differences and characterized their spatial distributions by using of differential gene expression analysis,enrichment analysis and Immunohistochemistry etc.In this research,we identified a group of SPARCL1~+fibroblast,which plays a role in protecting blood vessels from tumor cell invasion and is related to the favorable prognosis of liver cancer patients.Finally,we investigated the origin of these cells by performing pseudo-chronological analysis and constructing transformation models in vitro.The second part is investigation on susceptibilities of stomach for SARS-CoV-2.The coronavirus disease 2019(COVID-19)epidemic has now spread to more than 200countries or regions around the world and is an ongoing global health crisis.Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)mainly infects cells by binding to the cellular receptor protein(ACE2).Recent studies showed that 20–50%patients had gastrointestinal symptoms as initial symptoms,and a large number of patients would have GI symptoms during hospitalization,suggesting that SARS-CoV-2 could be transmitted via fecal-oral route.As the main organ of the upper gastrointestinal tract,the susceptibility of the stomach to SARS-CoV-2 is still lacking in corresponding research.To reveal the SARS-CoV-2 susceptibility of different cells in gastric tissue,we performed an integration of 12 normal gastric sc RNA-seq datasets and identified 12 cell subpopulations.Our results reveal that there is a special type of intestinal epithelial cells in the stomach tissue,which highly express ACE2.This type of cells is mainly derived from the intestinal metaplasia(IM)samples of the stomach.By calculating the intestinal metaplasia gene set expression,it was found that the IM index was significantly correlated with ACE2.As Helicobacter pylori(H.pylori)infection is an important risk factor for IM,it is speculated that H.pylori infection and intestinal metaplasia may be an important susceptibility factor for stomach infection with SARS-CoV-2.Subsequently,we analyzed the clinical and laboratory examination data of 420 patients with COVID-19 and found that the patients with upper gastrointestinal symptoms had a higher percentage of H.pylori infection than those with lower gastrointestinal symptoms and the application of gastric mucosal protective agents can significantly reduce the follow-up occurrence of diarrhea symptoms.This study described the distribution of SARS-CoV2 receptors in stomach,revealed the susceptibility factors of stomach for SARS-CoV-2,explored the mechanism of non-diarrheal GI symptoms in COVID-19 patients and provided important biological knowledge for guiding the prevention and treatment of COVID-19.