The Establishment Of Gut Microbiota Predictive Model Based On Microbiome Research And The Correlation With TME In HCC-MVI

Objective:The aim of this study is to investigate the characteristics of gut microbiota in hepatocellular carcinoma(HCC)patients with vascular invasion(HCC-VI),establish a microbial prediction model of HCC patients with microvascular invasion(HCC-MVI)preliminarily,analyze the changes of related indexes in tumor microenvironment(TME)of HCC-MVI,and explore the correlation between the TME and gut microbiota.Methods:Part I:A total of 75 study subjects were enrolled in the study from January 2020 to December 2020,including 54 males(72.0%)and 21 females(28.0%),with an average age of 56.4±10.5 years.Fecal samples were collected from healthy volunteers(n=16),HCC patients without vascular invasion(HCC-NVI)(n=20),HCC-MVI(n=24)and HCC patients with macrovascular invasion(HCC-Ma VI)(n=15).Genomic DNA of gut microbiota was extracted and sequenced on Illumina Nova Seq6000.The changes of gut microbiota in HCC group and HCC-VI group were analyzed by bioinformatics methods.The microbial prediction model of HCC-MVI was established based on random forest method.Part II:HCC-NVI(n=20)and HCC-MVI(n=24)who underwent liver resection in Part I were enrolled.Immunohistochemical staining and immunofluorescence staining were used to observed the percentage of CD8~+T cell,M1-type tumor associated macrophage(TAM)and M2-type TAM in TME on tumor paraffin sections,and the changes of HCC-MVI group were further analyzed.Spearman correlation and canary correlation analysis(CCA)were used to analyze the correlation between gut microbiota and related indexes of TME in HCC-MVI group.Results:Part I:(1)The structure of gut microbiota of HCC patients was significantly different from that of healthy control group.The diversity of gut microbiota in HCC-VI group was decreased,and the structure was significantly different from that of HCC-NVI group and healthy control group(all P<0.01);(2)The relative abundance of Bacteroidetes in HCC patients was higher,while the relative abundance of Firmicutes and Actinobacteria were lower than that in healthy control group at the phylum level.The relative abundance of Bifidobacteria was decreased,while the relative abundance of Prevotella and Klebsiella was increased than that in healthy control group below phylum level(all P<0.05);(3)The relative abundances of multiple gut microbiota in HCC-MVI and HCC-Ma VI group were decreased at the genus level.In general,the relative abundances of Firmicutes,Ruminococcus and Monoglobales in two groups were decreased compared with that in HCC-NVI group and healthy control group,while the relative abundance of Prevotella was increased significantly(all P<0.05);(4)The characteristic microbiota of gut was distinct in different vascular invasion groups.Based on the results of comparative analysis on microbial relative abundance between groups and LEf Se analysis,Klebsiella was increased significantly in HCC-MVI group compared with HCC-NVI group and healthy control group,which was the key differential microbiota of HCC-MVI patients,while Prevotella was increased significantly in HCC-Ma VI group compared with HCC-NVI group and healthy control group,which was the key differential microbiota of HCC-Ma VI patients;(5)The prediction model of HCC-MVI was established based on microbiology and the area under the ROC curve was 99.65%,which showed a good prediction efficiency.Part II:(1)Compared with HCC-NVI group,the percentages of CD8~+T cell and M1-type TAM in TME of HCC-MVI group were decreased(P=0.001;<0.001),while the percentage of M2 type TAM was increased(P<0.001);(2)Correlation analysis between gut microbiota and TME of HCC-MVI group showed that M2-type TAM was significantly negatively correlated with Shannon index and Simpson index(all P<0.01);(3)The metabolism functions of gut microbiota in HCC-MVI group were significantly concentrated in replication and repair,translation,energy metabolism,nucleic acid metabolism,and polysaccharide biosynthesis and metabolism;(4)Correlation analysis result between the functional pathway and percentage of M2-type TAM in HCC-MVI group showed that m TOR signaling pathway was positively correlated with percentage of M2-type TAM(r=0.37,P=0.01).Conclusions:In this study,for HCC-VI group,the diversity of gut microbiota was decreased and the structure of gut microbiota was significantly changed.Klebsiella was the key differential microbiota of HCC-MVI patients,while Prevotella was the key differential microbiota of HCC-Ma VI patients.The microbial prediction model of HCC-MVI had a good predictive efficacy which requires validation using large-sample,multicenter microbial datasets.Correlation analysis between structure and function changes of gut microbiota and related indexes of TME in HCC-MVI group based on Spearman correlation and CCA methods showed that the percentage of M2-type TAM was significantly negatively correlated with diversity of gut microbiota,and the m TOR signaling pathway might be involved in the increase of the percentage of M2-type TAM.This result provided a new idea to explore the malignant biological behaviors of HCC,which needed further investigation from mechanism.