| Microorganisms in human body are closely related to human health.Balanced microecosystem plays an important role in maintaining human health,and the imbalance of microecology may lead to the occurrence and development of diseases.At the same time,the disease state will affect the structure and composition of specific functional groups of human microorganisms,resulting in changes in the structure and diversity of microbial communities.Hepatocellular carcinoma is a disease that seriously threatens human life and health.Therefore,it is important to study the characteristics of microbial community of patients with hepatocellular carcinoma,to find the keystone taxa in the microbial community of patients,to explore the occurrence and development mechanism of hepatocellular carcinoma,and to evaluate its potential for clinical diagnosis and treatment.It plays an important role in early diagnosis and treatment of hepatocellular carcinoma patients and saving patients' lives.In this study,the theory and method of molecular ecology were used to study the composition,diversity and key groups of bacterial community in feces and blood from patients with hepatitis B cirrhosis and hepatocellular carcinoma,and from their healthy first-degree relatives,and its correlation with clinical physiological parameters was studied systematically.The purpose of this study is to clarify the construction mechanism of bacterial community in human microhabitat of patients with hepatocellular carcinoma,to clarify the relationship between the composition of blood and intestinal bacterial community,to explore the source of blood microbiota,and to provide to reliable biomarkers for early diagnosis of hepatocellular carcinoma.The specific results are as follows:(1)Characteristics of intestinal bacterial community in patients with hepatocellular carcinomaCluster analysis and ternary phase diagram analysis showed that the intestinal bacterial community structure of patients with hepatocellular carcinoma changed significantly at the family and genus level.The ? diversity of intestinal bacteria in AFPH group was significantly different from that in AFPL group(P<0.05).It suggested that the disease affected the bacterial community structure of human body,but the intestinal bacterial community structure and diversity caused by the level of AFP in patients with hepatocellular carcinoma were not the same.Two differential taxa,Romboutsia and Veillonella,were screened by Metastats,LEf Se and Random Forest analysis.ROC curve analysis showed that the Romboutsia,Peptostreptococcus,Romboutsia and Veillonella,Fusobacterium,Faecalibacterium,and Peptostreptococcus had higher diagnostic value in hepatocellular carcinoma.The analysis of RDA and Spearman methods showed that the key differential taxa between patients with hepatocellular carcinoma and healthy people were significantly correlated with a variety of physiological indexes of human(P<0.05).Through the construction of molecular ecological network,it was further confirmed that Firmicutes and Proteobacteria were the key groups of intestinal microbiota in patients with hepatocellular carcinoma.(2)Characteristics of blood bacterial community structure in patients with hepatocellular carcinomaThrough cluster analysis and ternary phase diagram analysis,the blood bacterial community composition of patients with hepatocellular carcinoma changed significantly at the family,genus and species level.Konellia,Thermomonas,Romboutsia and Veillonella were selected as the differential taxa of bacterial community in the blood of patients.Based on the ROC curve analysis,it was found that the combination of intestinal microbiota and blood microbiota could significantly improve the diagnostic accuracy.It was worth noting that the accuracy of Firmicutes in the diagnosis of hepatocellular carcinoma was as high as 0.939 for patients with AFPL,which was expected to provide a theoretical basis for AFPL patients suspected of HCC.The correlation between blood bacteria and human clinical physiological indexes in patients with hepatocellular carcinoma was analyzed by RDA and Spearman methods.It was found that the key differential taxa were significantly related to a variety of human physiological indexes(P<0.05),which might be involved in the pathophysiological process of the disease and the occurrence of hepatocellular carcinoma.It was clear that Firmicutes and Proteobacteria were the differential taxa of blood microbiota in patients with hepatocellular carcinoma.The keystone taxa in patients with hepatocellular carcinoma were less than those in healthy groups,and the keystone taxa could not be found in blood bacteria in patients with AFPH,indicating that the stability of bacterial community structure in patients with increased AFP was decreased.(3)The relationship between intestinal and blood microbial communitiesThe Venn analysis showed that vast majority of intestinal and blood bacteria were common bacteria,and only a small number of them were endemic to each other,no matter in the state of health or disease.Therefore,we speculated that most of the blood bacteria in patients with hepatocellular carcinoma might originate from the intestine through the gut-liver axis.There was a significant increase in the number of blood bacteria peculiar to patients with hepatocellular carcinoma,indicating that these increased blood bacteria might be related to the occurrence and development of hepatocellular carcinoma.Through the correlation analysis of bacterial community diversity in patients with hepatocellular carcinoma and healthy people,it was found that there was a negative feedback relationship between intestinal and blood bacterial diversity in healthy people,however,there was no significant correlation between the alpha diversity of the intestinal and blood bacterial of hepatocellular carcinoma patients.The similarity of microorganisms in the family was studied by the combination of cluster chart and histogram,Heatmap and Wilcoxon symbolic rank test of paired samples.The results showed that the intestinal and blood bacteria of patients and healthy people in the family were similar at the genus level.This might be caused by common genetic factors,geographical environment,living habits and diet.(4)The possible mechanism of microbial imbalance promoting the progression of hepatocellular carcinomaSome studies had shown that the imbalance of intestinal microbiota activates m TOR,and leads to the occurrence of hepatocellular carcinoma.In this study,q RT-PCR and Western blot were used to detect the expression of SNRPA1 m RNA and protein in hepatocellular carcinoma cells after silencing m TOR(P<0.05).The results showed that activating m TOR increased the expression of SNRPA1 m RNA and protein.Furthermore,the tumorigenicity of SNRPA1 in nude mice and the effect of SNRPA1 on the proliferation of HCC cells in vitro were studied by knocking down the SNRPA1 gene of hepatoma cells.The results showed that SNRPA1 gene could cause tumor in vivo and promote the proliferation of hepatoma cells.Therefore,it was speculated that dysfunctional intestinal bacterial may increase the expression of SNRPA1 by activating m TOR,and promote the occurrence and development of hepatocellular carcinoma.To sum up,the structure of intestinal and blood bacteria in patients with hepatocellular carcinoma changed significantly at the family,genus and species level.The physiological indexes of patients are significantly related to the structure of bacterial community.The key differential taxa of bacterial community alone or in combination might provide a theoretical basis for non-invasive diagnosis of hepatocellular carcinoma,and the results had important application value. |
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