Integrated microbiome and metabolome analysis reveals the interaction between intestinal flora and serum metabolites as potential biomarkers in hepatocellular carcinoma patients

Globally, liver cancer poses a serious threat to human health and quality of life. Despite numerous studies on the microbial composition of the gut in hepatocellular carcinoma (HCC), little is known about the interactions of the gut microbiota and metabolites and their role in HCC. We examined the composition of the gut microbiota and serum metabolic profiles in 68 patients with HCC, 33 patients with liver cirrhosis (LC), and 34 healthy individuals (NC) using a combination of metagenome sequencing and liquid chromatography‒mass spectrometry (LC‒MS). The composition of the serum metabolites and the structure of the intestinal microbiota were found to be significantly altered in HCC patients compared to non-HCC patients. We used LEfSe and metabolic pathway enrichment analysis to identify two key species ( Odoribacter splanchnicus and Ruminococcus bicirculans ) and five key metabolites (ouabain, taurochenodeoxycholic acid, glycochenodeoxycholate, theophylline, and xanthine) associated with HCC, which we then combined to create panels for HCC diagnosis. We discovered that the diagnostic performance of the metabolome was superior to that of the microbiome, and a panel comprised of key species and key metabolites outperformed AFP in terms of diagnostic value. Spearman’s rank correlation test was used to determine the relationship between the intestinal flora and serum metabolites and their impact on hepatocarcinogenesis and progression. A random forest model was used to assess the diagnostic performance of the different histologies alone and in combination. The panel we developed was very close to the AUC values of the best omics. In summary, this study describes the characteristics of HCC patients’ intestinal flora and serum metabolism, demonstrates that HCC is caused by the interaction of intestinal flora and serum metabolites, and suggests that two key species and five key metabolites may be potential markers for the diagnosis of HCC. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was supported by funding for infectious disease clinical center of Nanjing and infectious disease innovation center of Jiangsu ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics Committee of The Second Hospital of Nanjing gave ethical approval for this work. Lot No.2021-LY-ky051 I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors All data produced in the present work are contained in the manuscript All data produced are available online