High-throughput sequencing analyses of the correlation between hepatic echinococcosis infection and the ecological microenvironment of extrahepatic cholangiocarcinoma-like organs
Objective To investigate the effect of hydatid antigen treatment on the ecological microenvironment of extrahepatic cholangiocarcinoma(CCA)organoids by using 16S rRNA high-throughput sequencing technology and organoid models,and to reveal the potential role of hydatid cysts in the pathogenesis of CCA.Methods This study was a prospective controlled study,in which 60 patients diagnosed with extrahepatic cholangiocarcinoma(CCA)were enrolled and divided into an experimental group(exposed to hydatid antigen)and a control group(exposed to PBS),with 30 samples in each group.Tumor tissue samples were cultured by organoid technology,and 16S rRNA high-throughput sequencing technology was used to analyze the microbial community composition and functional gene abundance of the two groups of samples.The linear discriminant analysis effect size(LEfSe)method and PICRUSt tool were used for microbial community difference analysis and functional prediction analysis,and the changes of key microbial groups and signaling pathways were further verified by qPCR and cell function experiments.Results The main microbial phyla in the experimental and control groups included Firmicutes,Bacteroidetes,Proteobacteria,Actinobacteria,and Thermotogae.The abundance of Firmicutes and Actinobacteria in the experimental group was significantly higher than that in the control group,while the abundance of Bacteroidetes was significantly decreased(P＜0.05).α-diversity analysis showed that the Chao1 index(150.25±15.75)and Shannon index(3.50±0.45)in the experimental group were significantly higher than those in the control group(Chao1 index:140.80±14.95,Shannon index:3.31±0.40,P＜0.05).The results of principal coordinates analysis(PCoA)and nonmetric multidimensional scaling(NMDS)based on Bray-Curtis distance for β-diversity analysis showed that there were significant differences in the composition of the microbial communities between the experimental and control groups(PERMANOVA:F=2.423,P＜0.001).PICRUSt analysis results showed that the gene abundance of the experimental group in functional pathways such as carbohydrate metabolism(25.30％±2.50％vs 23.10％±2.40％),energy metabolism(18.75％±1.95％vs17.50％±2.10％),lipid metabolism(15.20％±1.80％vs 14.00％±1.75％),amino acid metabolism(12.50％±1.60％vs 11.20％±1.55％),nucleotide metabolism(10.00％±1.25％vs 9.00％±1.20％)and signal transduction(8.75％±1.10％vs 7.50％±1.05％)was significantly higher than that in the control group(P＜0.05).qPCR results showed that the relative abundance of Firmicutes(t=2.191,P=0.033),Proteobacteria(t=2.253,P=0.029)and Actinobacteria(t=2.311,P=0.026)in the experimental group increased significantly,while the relative abundance of Bacteroidetes(t=2.432,P=0.021)and Thermotolerant bacteria(t=2.732,P=0.009)decreased significantly.Cell function experiments showed that the experimental group had significantly enhanced cell proliferation,migration and invasion abilities(P＜0.05).Spearman correlation analysis showed that the abundance of Firmicutes was significantly positively correlated with patient age(r=0.452,P=0.001)and medical history(r=0.322,P=0.013),and was significantly negatively correlated with tumor grade(r=-0.455,P=0.001),and was significantly positively correlated with tumor stage(r=0.311,P=0.024).Conclusion This study revealed the significant effect of hydatid antigen treatment on the microecological environment of extrahepatic cholangiocarcinoma organoids through high-throughput sequencing and functional prediction analysis,especially in the changes of microbial community composition and functional gene abundance.
Hepatic echinococcosisextrahepatic cholangiocarcinomaorganoids16S rRNA high-throughput sequencingmicrobial community
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