Diversity Of Gut Flora In Patients With Cholangiocarcinoma By High-Throughput Sequencing

Objective:Intestinal flora play important roles in the pathogenesis of digestive system malignant tumors.However,the role of intestinal microflora in the pathogenesis of cholangiocarcinoma is still unclear.In this study,the species composition and diversity of intestinal flora in patients with cholangiocarcinoma and healthy people were analyzed by16 SrDNA high-throughput sequencing,and the intestinal microecological changes of patients with cholangiocarcinoma were analyzed to find the potential correlation between cholangiocarcinoma and intestinal flora,so as to provide a theoretical basis for exploring the correlation between the etiology;pathogenesis of cholangiocarcinoma and intestinal flora.Methods:A total of 20 patients with cholangiocarcinoma in the Gastroenterology Medicine Department and Hepatobiliary Surgery Department of Qingdao Municipal Hospital Affiliated to Qingdao University from August 2018 to August 2019 were selected.20 healthy volunteers undergoing physical examination at the same time were selected as the control group.The feces samples of the two groups were collected for DNA extraction and amplification by PCR,and the products were purified,quantified and homogenized to form a sequencing library.Based on Illumina HiSeq2500 sequencing platform and the method of double-terminal sequencing,a small fragment library was constructed for sequencing to reveal species annotation and abundance analysis,the species composition of intestinal flora in the two groups through Reads splicing,filtering,OTUs clustering.And α diversity,β diversity and species differences between groups were analyzed.Results:1.A total of 2534941 effective sequences were obtained in the two groups,including1272740 in the cholangiocarcinoma group and 1262201 in the control group.2.At the phylum level,Firmicutes,Bacteroidetes and Proteobacteria had high abundances in both groups,and their abundances in cholangiocarcinoma group were30.66%,20.67% and 19.21%,respectively,and those in the control group were 59.84%,12.80% and 13.30%,respectively.3.At the genus level,the relatively abundant bacteria in the two groups were Blautia,Bacteroides,Faecalibacterium,Escherichia-Shigella,uncultured＿bacterium＿f＿Muribaculaceae and so on.The relative abundances in the cholangiocarcinoma group and the control group were 3.33%,3.05%,1.12%,1.30%,4.97% and 8.06%,8.25%,8.77%,6.07%,0.28%,respectively.4.In terms of Alpha diversity,the average value of Chao1 in cholangiocarcinoma group was 806.63 ± 83.31,while that in control group was 552.99 ± 153.72;the average value of Ace in cholangiocarcinoma group was 794.98 ± 76.09,while that in control group was593.82 ± 148.73,suggesting that the species abundance of intestinal flora in cholangiocarcinoma group was significantly higher than that in control group(P<0.05).The average Shannon index of cholangiocarcinoma group was 5.24 ± 0.32,while that in control group was 3.55 ± 0.71;the average value of Simpson index in cholangiocarcinoma group was 0.019 ± 0.017,while that in control group was 0.0745 ±0.046,suggesting that the Alpha diversity of intestinal flora in patients with cholangiocarcinoma was significantly higher than that in healthy people(P<0.05).5.In terms of β diversity analysis,the species diversity of intestinal flora in the two groups was compared based on PCA analysis,PCoA analysis,NMDS analysis and other methods.The results showed that there were significant differences in species diversity between the two groups.6.In terms of significant difference between groups,the relative abundance of Bacteroidetes,Acidobacteria and Verrucomicrobia in intestinal flora of patients with cholangiocarcinoma was significantly higher than that in the control group,while Firmicutes and Actinobacteria were significantly lower than those in the control group.At the genus level,the relative abundance of Faecalibacterium,Blautia,Bifidobacterium and Bacteroides in the intestinal flora of patients with cholangiocarcinoma was significantly lower than that in the control group,while the relative abundance of Acinetobacter and Helicobacter in the intestinal flora of patients with cholangiocarcinoma was significantly higher than that in the control group(P<0.05).Conclusion:There are significant differences in species abundance and diversity between patients with cholangiocarcinoma and healthy people,and the imbalance of intestinal flora may play an important role in the pathogenesis of cholangiocarcinoma.