| Background:Lung cancer is a high-incidence,high-mortality and high-burden neoplastic disease worldwide.The overall survival rate of lung cancer is still very low,due to the late diagnosis and resistance to standard chemotherapy.In the past 50 years,the survival rate of lung cancer has hardly improved.Thanks to significant advances in targeted therapy and immunotherapy for lung cancer,we need to have a deeper understanding of the local environment of lung cancer to improve the diagnosis and treatment of individualized lung cancer.The symbiotic microbiota can provide the body with nutrients or disease susceptibility through a variety of ways.At present,we have found that in many systemic diseases such as diabetes,obesity,chronic gastritis and colorectal cancer,the symbiotic balance between human body and microorganisms is broken.New research evidence suggests that the ecological imbalance of microbiota may affect the occurrence and development of cancer through multiple level pathways(such as by affecting metabolism,inflammation or immune pathways).Epidemiological evidence suggests that there is a relationship between repeated exposure to antibiotics and increased risk of lung cancer,but the role of lung's microbiome in lung cancer remains unclear.Previously,healthy lungs were considered to be sterile,but current research indicates that there is a microbial community in the lungs,and that the microbiome has changed in respiratory diseases including asthma,chronic obstructive pulmonary disease(COPD),and cystic fibrosis.Whether the pulmonary microbiome affects the development of lung cancer,some related research has been carried OTU.Epidemiological studies suggest that Mycobacterium tuberculosis is an important risk factor for lung cancer.Studies have also shown that lung cancer patients have important changes in the structure of the microbiome,such as the increase in the abundance of bacterial genera or phylum,but the overall ? diversity is reduced.However,further correlations between lung cancer and the bacterial flora are still unknown.Therefore,we have designed this research.OBJECTIVE:To explore the overall structural characteristics and comparative analysis of upper and lower respiratory tract microbiomes in patients with lung cancer,to explore the metabolomic changes in BALF of lung cancer patients,to screen potential markers related to lung cancer,and to explore the relationship between pulmonary genus and metabolites.Method:We collected pharyngeal swabs,bronchoalveolar lavage fluid(BALF),cancerous and non-cancerous lung tissue samples,and used Illumina MiSeq high-throughput second-generation sequencing technology platform and bioinformatics analysis platform to analyze the V3-V4 region of 16 S rDNA gene in samples of bacteria to explore the overall structural characteristics of upper and lower respiratory tract microbiome in lung cancer patients.At the same time,we have studied the BALF of lung cancer patients by gas chromatography-mass spectrometry(GC-MS)and metabolomics research methods,hoping to screen and excavate potential markers related to lung cancer.Based on the above two studies,combined with changes in microbiome and metabolites in lung cancer patients,the relationship between lung bacteria and metabolites was initially explored.Result:??Characteristics of respiratory microbiome and comparative study results in healthy people,non-lung cancer lung diseases and lung cancer patients(?)?Structural characteristics of pharyngeal microbiome in patients with lung cancer(35 cases of pharyngeal swabs: 11 healthy people,9 non-lung cancer lung diseases,15 lung cancer patients)1?The pharyngeal microbiome mainly includes four dominant phylums: Bacteroidetes,Firmicutes,Proteobacteria and Fusobacteria;nine dominant genus: Prevotella,Veillonella,Neisseriales,Streptococcus,Haemophilus,Fusobacterium,Alloprevotella,Leptotrichia,Porphyromonas.2?Comparing the three groups,the ? diversity of lung cancer patients was reduced.Comparing the abundances of the three groups,the abundance of the Firmicutes in lung cancer and non-lung cancer lung diseases patients was significantly increased,and Fusobacteria was significantly reduced in lung cancer patients;Veillonella is significantly elevated in lung cancer patients and non-lung cancer patients.Streptococcus is mainly elevated in lung cancer patients.Alloprevotella is significantly reduced in lung cancer patients and non-lung cancer patients,Actinomyces and Rothia are reduced in patients with non-lung cancer disease.In addition,in some less abundant genus,the abundance of Pseudomonas,Oribacterium,Kingella and Lachnoanaerobaculum in lung cancer patients and non-lung cancer lungs was significantly reduced.3?Among the rare genus,compared with the other two groups,the genus Parvimonas,Lactobacillus,Peptostreptococcus,Solobacterium,Peptococcus,and Bulleidia increased significantly.ROC diagnostic analysis of lung cancer patients and non-lung cancer patients(healthy and non-lung cancer patients)by 6 genus with significant differences,found that three genus:Parvimonas,Solobacterium and Peptococcus has a high diagnostic value for the identification of lung cancer.(?)?Structural characteristics of BALF microbiome in patients with lung cancer(83 BALF samples: 20 healthy people,25 non-lung cancer lung diseases,38 lung cancer patients)1?The six dominant phylums in BALF are: Firmicutes,Bacteroidetes,Proteobacteria,Fusobacteria,Actinobacteria,and Spirochaetes;The dominant nine major genus are: Prevotella,Streptococcus,Veillonella,Haemophilus,Neisseriales,and Alloprevotella,PorPhyromonas,Fusobacterium,Actinomyces,Campylobacter,Treponema,pseudomonas Pseudomonas and Lactobacillus.2?Compared with the three groups,the ? diversity of lung cancer patients and patients with non-lung cancer diseases decreased.Comparison of microbiome abundance between the three groups: the abundance of Firmicutes and Actinobacteria in lung cancer patients and non-lung cancer patients was significantly increased,and Fusobacteria is significantly less abundant in patients with non-lung cancer disease;Streptococcus and Veillonella are significantly elevated in lung cancer patients and non-lung cancer disease patients,and Alloprevotella is significantly reduced in non-lung cancer lung diseases.Fusobacterium has a markedly reduced abundance in lung cancer patients and non-lung cancer disease patients.Comamonas is reduced in lung cancer patients and elevated in non-lung cancer disease patients.In addition,in some less abundant genus,Pseudoramibacter,Pyramidobacter,Pyramidobacter,Sphaerochaeta,Sphingomonas,Deinococcus are elevated in lung cancer patients.The abundance of Acinetobacter is reduced.3 ? Among the rare genus,Pseudoramibacter,Chryseobacterium,Acinetobacter,Comamonas,Pyramidobacter,Sphaerochaeta,Sphingomonas,Leucobacter,Deinococcus abundance Significantly elevated in patients with lung cancer.The ROC diagnostic analysis of lung cancer patients and non-lung cancer patients(healthy and non-lung cancer patients)was carried OTU using 10 genus with significant differences.It was found that Sphaerochaeta and Comamonas have higher diagnostic value for identifying lung cancer.4?A subgroup comparison of patients with adenocarcinoma and squamous cell carcinoma showed that the ? diversity of squamous cell carcinoma patients decreased,there was no significant difference between the main phylums abundances,but,Haemophilus was found Significantly elevated in patients with squamous cell carcinoma.(?)?Comparative analysis of the structure of upper and lower respiratory microbiome in three groups of self-control1?The lower respiratory tract microbiome OTU of healthy people overlaps with the upper respiratory tract flora OTU by 92%,the lower respiratory tract microbiome OTU of lung cancer patients overlaps with the upper respiratory tract flora OTU by 95%,and the lower respiratory tract microbiome OTU and upper respiratory tract flora of non-lung cancer diseases OTU overlaps 82%.It is suggested that the majority of the lower respiratory microbiome in the three groups may be derived from the upper respiratory tract.In addition,from the trend of change,the lower respiratory tract microbiome of lung cancer is more susceptible to the upper respiratory tract.2?Comparative trend analysis of ? diversity: The difference between up and lower respiratory microbiome ?diversity in patients with non-lung cancer disease is reduced,while the difference between up and lower respiratory microbiome ?diversity in lung cancer patients increases;In patients with non-lung cancer lung disease and lung cancer,between the upper and lower respiratory tract the difference of Bacteroidetes/ Actinobacteria's abundance is reduced,It is indicated that the two phylums in the lower respiratory tract are susceptible to the upper respiratory flora.n lung cancer patients,the difference between the upper and lower respiratory tracts of Proteobacteria and Fusobacteria increases.It is indicated that the two types of genus in the lower respiratory tract are less affected by the upper respiratory tract in lung cancer patients.The difference between the upper and lower respiratory tracts of the Firmicutes in lung cancer disappeared.This indicates that in patients with lung cancer,Firmicutes are more susceptible to the upper respiratory tract microbiome.In non-lung cancer disease and lung cancer patients,the difference in abundance of Porphyromonas/Actinomyces between the upper and lower respiratory tract is reduced.It is indicated that the influence of the pharyngeal microbiome is increased in the lower respiratory tract for Porphyromonas and Actinomyces.In patients with lung cancer,Neisseria has an increased difference between the upper and lower respiratory tract,indicating that two genus of the lower respiratory tract are less affected by the pharyngeal microbiome in lung cancer.The difference between the upper and lower respiratory tract of Prevotella and Streptococcus disappeared in lung cancer,indicating that the two species of lung cancer are more susceptible to the upper respiratory tract microbiome.??Characteristics and comparative study results of lung cancer tissue and non-lung cancer tissue(?)?A sample of 19 lung tissue samples was collected for microbiome analysis.There were 5 cases of "healthy" lung tissue,14 cases of lung adenocarcinoma(5 cases of proximal leaf bronchus,5 cases of adenocarcinoma,4 cases of distal alveolar tissue)(?)Analysis of structural characteristics of main microbiomeThe five major phylums in lung tissue samples include: Proteobacteria,Bacteroidetes,Firmicutes,Acidobacteria,and Actinobacteria.Compared with "healthy" tissues,lung cancer tissues showed a significant decrease in ? diversity.Compared with proximal airway tissue and distal alveolar tissue,the ? diversity was significantly reduced.The abundance of Bacteroidetes,Acidobacteria and Actinobacteri was increased in lung cancer patients,and the abundance of Firmicutes was decreased,while there was no significant difference in the tissues of three parts of lung cancer patients.In the lung cancer patients,the abundance levels of Sediminibacte,Ralstonia,Rhodanobacter,Xanthomonas and Delftia increased,and the abundance levels of Lactobacillus and Caulobacter decreased,However,there was no significant difference in the abundance of the tissues between the three sites of lung cancer patients.??BALF metabolomics study results in patients with lung cancer(?)?The number of significant differential metabolites screened for lung cancer patients was 35(14 of which decreased and 21 increased).Major disorders of metabolic pathways include alanine,aspartate and glutamate metabolism,glycine,serine and threonine metabolism,aminoacyl-tRNA biosynthesis,pentose phosphate pathway,glycolysis or gluconeogenesis Pyruvate metabolism.(?)?10 differential metabolites with AUC greater than 0.9 were screened,and 10 metabolites with potential predictive value were: xanthine,2-aminobutyric acid,glucose,isoleucine,valine,leucine,glutamic acid,proline,alanine,and tryptophan.A combined diagnosis of 7 amino acids and ROC analysis of lung cancer,combined 7 amino acids to predict the area under the ROC curve of lung cancer was 0.975,sensitivity was 88%,specificity was 100%.??Correlation between BALF microbiome and metabolome in patients with lung cancerBased on the first part and the third part of the study,the correlation analysis between the differential metabolites and the genus of BALF in lung cancer patients was analyzed by linear correlation coefficient.The results showed that: Proline,Alanine,Methionine,Ornithine,Glucose,2-aminobutyric acid and threonine are negatively correlated with Lactobacillus;2-stearoylphosphate,2-palmitoylglycerol,and 2-hydroxypyridine are negatively correlated with Lactobacillus;Cysteine,xanthine,Proline,Alanine,Methionine,Ornithine,Glucose,2-aminobutyric acid and threonine are positively correlated with Comamonas;palmitic acid,uracil,2-stearoylphosphate,2-palmitoylglycerol,and 2-hydroxypyridine are negatively correlated with Comamonas;Actinomyces is positively associated with Proline and Ornithine;negatively correlated with palmitic acid;Pseudomonas is positively correlated with Methionine;Fusobacterium is positively correlated with uracil.It suggests that the structural changes of lung microbiome in lung cancer patients may affect these metabolites and metabolic pathways in the lung,which in turn affects the host's metabolic phenotype.Conclusion:we used the Illumina MiSeq high-throughput second-generation sequencing technology platform and bioinformatics analysis platform to analyze the throat of the Han people by analyzing pharyngeal swab samples from healthy people,non-lung cancer patients with lung diseases and lung cancer patients.The pharyngeal microbiomes mainly includes 4 major phylums and 9 genus.The alpha diversity of the pharyngeal microbiomes of lung cancer patients will decrease,and the relative abundance of some bacteria and bacteria will increase,while others will decrease.By analyzing the samples of BALF in healthy people,non-lung cancer patients with lung diseases and lung cancer patients,it is found that the BALF microbiomes of Han people mainly includes 6 major phylums and 9 genus.The diversity of BALF microbiomes in lung cancer patients decreased,and the relative abundance of some phylums and genus increased,while others will decreased.Among them,Comamonas and Sphaerochaeta may be important for identifying lung cancer.In addition,squamous cell carcinoma patients have reduced alpha diversity compared with adenocarcinoma.The self-matching study compared the upper and lower respiratory tracts and found that the lower respiratory tract microbiomes mostly originated from the upper respiratory tract,and the lower respiratory tract microbiomes was affected by the upper respiratory tract microbiomes in different disease states.By analyzing the lung cancer tissue and non-lung cancer tissue microbiomes,it is found that the lung tissue samples mainly include 5 major phylums and 7 genus,and the alpha diversity of lung cancer tissues is significantly reduced.We used gas chromatography-mass spectrometry(GC-MS)to analyze alveolar lavage fluid samples from healthy people and lung cancer patients using metabolomics research methods.We have learned that multiple metabolic pathways are dysfunctional in lung cancer patients,and we have screened for multiple potentially predictive metabolites.Finally,a multi-omics joint analysis strategy was applied to combine microbiome and metabolomics correlation analysis.We have learned that the metabolites of local disorders in lung cancer patients are positively correlated with Lactobacillus and Comamonas,and it is initially recognized how the structural changes of the microbiomes affect the local metabolic characteristics of lung cancer. |
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