Gut Microbiome Predicts The Prognosis Of Hepatocellular Carcinoma

Aims:Liver cancer is the fourth-most malignant tumor in China with the second mortality rate.Hepatocellular carcinoma accounts for 85%of all liver cancer cases.Hepatitis B virus infection is currently the leading cause of hepatocellular carcinoma.The gut microbiota has a symbiotic relationship with the organism,and plays a key role in many physiological and pathological processes.The gut-liver axis plays a role in the pathogenesis of hepatocellular carcinoma;however,the correlations between the gut microbiome and the liver tumor transcriptome in patients and the impact of the gut microbiota on clinical outcome are less well-understood.The recurrence after surgical resection severely restricts the long-term survival of patients;therefore,identifying gut microbial markers and establishing an early warning system for the recurrence/prognosis of hepatocellular carcinoma are highly desirable.Methods:Our team’s previous research obtained the gut microbiome data of 131 healthy volunteers and 150 hepatitis B-related hepatocellular carcinoma patients from Eastern China.These participants received revisits in September 2019.According to the exclusion criteria,113 patients and 100 healthy volunteers were identified as the research objects of this project.The principles and tools of quantitative ecology and bioinformatics were used to describe the characteristics and differences of gut microbiome between healthy volunteers and patients,and between patients with different tumor burdens.After a rigorous screening process,32 paired tumor and adjacent non-tumor liver tissues from 113 patients were subjected to RNA-seq.The local standard sequencing analysis obtained the transcriptome information of these 32 patients.The Pearson correlation was used to assess the correlation between gut microbiome and tumor transcriptome of 32 patients,and the correlation between the clinicopathological characteristics of 113 patients and gut microbiome.Immunohistochemistry was used to detect the protein expression levels of microbial-related differential genes in tumors.DAVID and Metascape were used to evaluate the signal transduction and molecular networks;CIBERSORTx and independent databases were used to evaluate the microbe-related tumor immune microenvironment.Random Forest and Support Vector Machine models were used to verify the ability of specific microbial biomarkers and to predict the overall survival and disease-free survival of 113 patients with hepatocellular carcinoma.Results:Bacteroides,Lachnospiracea incertae sedis and Clostridium XIVa were enriched in gut of the patients with high tumor burdens.By integrating the microbiome and transcriptome,OTU₀002 and OTU₀409 from Bacteroides,OTU₀134 and OTU₁149 from Lachnospiracea incertae sedis,OTU₀033 and OTU₀794 from Clostridium XIVa were negatively correlated with 29 genes from host tumor transcriptome,forming 31 OTU-gene dyads.The 29 microbial-related genes not only meet the criteria for differential genes,but also represent tumor suppressor genes for good clinical prognosis.The changes in transcription levels of these 29 genes reflected the positive activation of anti-tumor immune microenvironment,which is mainly reflected in thymus-dependent lymphocytes and natural killer cells.Bile acids were identified as important communication mediators between gut microbiota and host transcriptome.Finally,among above three genera,based on six microbial markers associated with tumor immune microenvironment or bile acid metabolism,for five-year overall survival,the average AUC of Random Forest was 81%and the average AUC of Support Vector Machine was 70%,for two-year disease-free survival,the average AUC of Random Forest was 68%,and the average AUC of Support Vector Machine was 70%.Conclusions:The changes in tumor immune microenvironment caused by the gut microbiota via serum bile acids may be important factors associated with tumor burden and adverse clinical outcome.Gut microbes can be used as biomarkers of clinical features and outcomes,and the microbe-associated transcripts of host tumors can partly explain how gut microbiota promotes the pathogenesis of hepatocellular carcinoma.