| Esophageal cancer is one of the most common gastrointestinal maligna ncies,and the death toll of esophageal cancer has been high in China for a long time.In February 2017,the latest data of national cancer center reported that ten thousand people were diagnosed with cancer,about 7 people in a minute in China in 2013.The incidence of esophageal cancer in all malignant tumor was 27%,mortality about 20% occupied the fourth.According to Global cancer statistics 2012,the number of deaths from esophageal cancer in the world is 40.02 million,of which the number of Chinese esophageal cancer deaths is 19.75 million,accounting for 49.35 percent of the Global total.The research scholar in esophageal cancer etiology believed that the occu rrence of esophageal cancer is affected by complex process include many factors,multi-stage,multiple genes,but exact cause is still not determined.In addition to,living environment is also the important factors influencing the occurs of esophageal cancer except the factors such as heredity,life behavior,eating habits et al..In recent years,some chronic infection were found for cancer risk factors,because of the H.pylori human papilloma virus(HPV)Epstein barr virus 3 and hepatitis b virus infection were carcinogenic.According to new research progress in recent years,the structural abnorm alities of intestinal flora is likely to be the direct cause of metabolic diseasecau sed by the improper diet just like obesity,high blood pressure,diabetes,coron ary heart disease and stroke.Some analyses from researchers indicate the colo rectal cancer is closely related to intestinal flora.In addition to,some reports have pointed out that the anormal flora structure of the digestive tract can cause esophageal persistent chronic inflammation,that can increase the risk of esophageal cancer 2-40 times,and the Barrett's esophagus and stomach disea se,increase the risk of esophageal cancer 30-400 times.In our research we will explore the association between esophageal cancer and flora structure of pharynx and feces specimens according to the microorganism composition ana lyzed by the gene sequencing technology and 16 s rDNA gene classification.Objective: To explore preliminary on the composition of pharynx and feces specimens in esophageal cancer patients.Methods: Microbial DNA was extracted from faeces samples and throat swab of esophageal cancer patient and normal people,and divided into four groups(ES group,esophageal cancer group pharynx swab.EF group,esophageal can cer faeces group.NCS group,normal control pharynx swab group.NCS group,normal control faeces group),respectively,then the 16 S rDNA V3-V4 region of the Eukaryotic ribosomal RNA gene were amplified by PCR.Purified ampli cons were sequenced on Illumina Hiseq2500.The raw reads were deposited into the NCBI Sequence Read Archive(SRA)database.Effective Tags were obtained through Quality control and reads assembly include Reads filtering,Reads assembly,raw tag filtering and Chimera checking and removal.The effective tags were clustered into operational taxonomic units(OTUs),then the representative sequences were classified into organisms and analyzed Alpha diversity and Beta diversity.Finally,we analyzed the taxa difference microorganism group by establishing the taxa annotation and species classify cation tree.Result: The effective tags obtained by the data processing of all specimens comply with subsequent analysis requirements.1.The data processingThe sequence number of each sample in four groups was 54567.05±7841.034 on average,after data filtering and Tags Splice.We got 14289161 effective sequences finally,and the maximum and minimum sequence number was 75817 and 39084,respectively.The average length of the total sequence was 487 bp on average.The effective tags obtained number was 55366.42±6154.67 in ES group,53756.92±10952.46 in EF group,54827.00±7486.13 in NCS group,53310.33±4480.43 in NCF group.The effective rate was 81.48±6.3%?84.62±1.90%?86.29±2.34%?85.34±0.42 %respectively.The OUT repeated 805 between ES group and NCS group,510 between EF group and NCF group.2.The species taxonomy analyzeThe species richness and distribution of species were more homogeneous in species annotation and species classification tree.There were obvious aggregation in groups and significant differences between groups.3.The diversity analyzeThe Chao1,Shannon index of Alpha diversity analysis were slightly higher between patient group than the normal population control group,but the difference were not significant(P>0.05).The rarefaction curve,shannon rarefaction curve and Rank Abundance curve illustrate that sequencing depth covers basicly the samples of all the species and the depth and amount of sequencing tends to saturation,abundance of sample is high,the bacterial flora of sample distribute uniformly.The boxplot shows that the samples of each group have better accumulation.The Non-metric multidimensional scaling of Beta diversity analysis showed that each group was separate,and samples of each group presented obvious tendency of gathering respectively.It showed that there were certain differences between groups on microflora structure,and obviously similarity between samples of each group.4.The species differencesAt the Phylum level,the analysis of species differences showed that 4 of total 21 were different in pharyngeal microflora include Spirochaetae,Tenericutes,Bacteroidetes,and Synergistetes,and 5 of total 17 in fecal samples include Firmicutes,Proteobacteria,Fusobacteria,Saccharibacteria and Actinobacteria.At the Genus level,26 of total 209 were different in pharyngeal micro flora,and 23 of 241 in fecal samples mainly include Firmicutes,Bacter oides,Prevotella,Rothia,Streptococcus,Filifactor,Veillonella,Peptostre ptococcus,Treponema,Rikenellaceae et al.,and some unclassified Genus.Conclusion: There was no difference in the abundance of microflora between patients with esophageal cancer and normal population,but there were significant differences in the distribution of microflora structure.The 26 genus of pharynx and 23 genera of faeces were different.It suggested that the microflora structure of the pharyngeal and faeces specimens of esophageal cancer patient was somewhat changed. |
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