| Lung cancer is one of the most common respiratory malignancies in China.The death toll of lung cancer in China has remained high for a long time.In 2017,the National Cancer Center of China pointed out that the incidence of malignant tumors in China has reached 270.59/100 thousand while the mortality rate was 163.83/100 thousand.Among them,lung cancer was the leading cause of morbidity and mortality throughout the country.Among the lung cancer,the main histological type was non-small cell lung cancer,which occupied about 80%-85% of the total number.It is reported that the incidence of lung cancer not only has a strong relationship with the abnormal expression of oncogene and/ or suppressor gene and the abnormal expression of protein and microRNA,but also with the smoking history,environmental factors and behavior.The disturbance of the environment in the body also plays a promotional role in the development of tumor.Studies have shown that,intestinal microflora plays an important role in the immune function of the host,metabolic processes,hormone regulation and so on.Studies like Kahn found that the bacterial surface lipopolysaccharide in intestinal microbes would combine with the surface of cerebral vascular endothelial cells' TLR4 receptor,thus activating the molecular pathway of intracranial cavernous hemangioma.Many studies have shown that the intestinal microbial disorders play an important role in the occurrence,development and therapeutic application of non-alcoholic liver cancer,prostate cancer and colorectal cancer.Therefore,the further study of intestinal microbes may be helpful to clarify the occurrence and development mechanism of tumor as well as to discuss the new thoughts of the treatment of tumor patients.The latest research showed there was a strong relationship between intestinal flora and lung cancer.It was also reported that abnormal flora structure of digestive tract would cause persistent chronic inflammation of lung and trachea as well as increase the risk of lung cancer by 2-40 times.In this study,gene sequencing techniques and 16S-rDNA gene classification were used to analyze the microbial composition in fecal excreta and pharynx secretion,and to explore the relationship between lung cancer patients and their intestinal and pharyngeal microbial structures.Objective: To explore preliminary on the composition of throat swab and feces specimens of lung cancer patients.Methods: After extracting the microbial DNA respectively from faeces samples and throat swab of lung cancer patients and normal people,the DNA was divided into four groups,lung cancer group pharynx swab(LS),lung cancer faeces group LF group(LF),normal control pharynx swab group(CS)and normal control faeces group(CF),with 12 cases per group.Then,the 16 S rDNA sequencing was analyzed.The PE250 model of Hiseq2500 was sequenced on machine by constructing the sequencing library according to the official description of Illumina.After removing the sequence with high N ratio,the low-quality sequence,the tags splicing and filtering,and finally the chimeric sequence of Tags,the effective data can be obtained.After the abundance statistics of Effective Tags,the UTO analysis on it was carried out.Then the diversity of microorganism Alpha was analyzed.Finally,Species classification tree was built and species were annotated to analyze the species diversity.Result: The effective tags obtained by the data processing of all specimens met with subsequent analysis requirements.1.The data processingFilter the raw data that was obtained after sequencing on the machine,including Tags splicing,Tags filtering and Tags chimerism.And a total of 2291816 valid sequences were obtained from these 42 samples.The average number of sequences per sample was 54567.05 ±7841.034,the maximum number of sequences was 76178 / sample,while the minimum number of sequences was 41458 / sample.The coverage of sequencing is good and the average length of the total sequence was 461 bp.OTU analysis showed that there were 366 repeats between the 1351 OUT of lung cancer patients' pharyngeal flora(group LF)and 1351 OTU of normal people's pharyngeal flora(group CF).Besides,there were 457 repeats between the 1259 OTU of fecal specimens of lung cancer patients' flora(LS group)and the 805 OTU of normal people's flora(CS group).2.The diversity analyzeThe Chao1 and Shannon index of Alpha diversity analysis of the patient group(EF,ES)were slightly higher than that of the normal population control group(NCF,NCS),but the difference were not significant(P>0.05).The rarefaction curve,shannon rarefaction curve and Rank Abundance curve illustrated that the sequencing depth covers basically all the species of the samples.The depth and amount of sequencing tended to saturation,abundance of sample was high,and the bacterial flora of sample distributed uniformly.Principal coordinate analysis(PCoA: principalpal CO-ordinates analysis)showed that the two groups of samples had a distinct trend of separation,indicating that there were some differences in the pharyngeal flora structure between the two groups.However,the two groups showed obvious aggregation tendency,which meant the similarities between the two groups was obvious.3.The species taxonomy analyzeThe species richness and distribution of samples were more homogeneous in species annotation and species classification tree.There was obvious aggregation within groups and significant difference between groups.4.The species differencesAt the Phylum level,the analysis of species differences showed that 4 of total 21 were different in pharyngeal microflora,including Spirochaetae,Tenericutes,Bacteroidetes,and Synergistetes.And 5 of total 17 in fecal samples,including Proteobacteria,Firmicutes,Fusobacteria,Actinobacteria and Saccharibacteria.At the Genus level,26 of total 209 were different in pharyngeal micro flora,and 23 of 241 in fecal samples,mainly including Firmicutes,Bacter oides,Prevotella,Rothia,Streptococcus,Filifactor,Veillonella,Peptostre ptococcus,Treponema,Rikenellaceae etc.and some unclassified Genus of bacteria.Conclusion: There were few differences in the abundance of microflora between the lung cancer patients and normal population,but the differences in the distribution of flora structure were significant.The 26 genus of pharynx and 41 genera of faeces were different.It suggested that there were some differences in the microflora structure of the pharyngeal and faeces specimens of lung cancer patients. |
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