| Background:Lung cancer(LC)is the main cause of death from cancer worldwide and its incidence has continued to rise in recent years.According to Global Cancer Statistics 2020,more than 30%of all newly diagnosed lung cancers and approximately 40%of global cancer-related deaths occurred in China,resulting in a high disease and socioeconomic burden.Given that the pathogenesis of LC is lack of systematically characterized,it is essential to explore the etiology of LC,which underscore the need for continued efforts to elucidate the pathogenesis of LC.Major functions of the gut microbiota include metabolic activities,trophic effects,immunity and protection of the colonized host against invasion by alien microbes.With the development of high-throughput sequencing technology,the role of the gut microbiota is being increasingly accepted and several researches indicated the influence of gut microbiota in different diseases with a particular emphasis on cancers.Recent evidences have shown that changes in the gut microbiota could influence immunity in distal tissues,which supported that the alterations in the composition of the gut microbiota may lead to systemic immune responses and diseases.An increasing number of studies have considered the potential mechanism of the gut microbiota in lung immunity and diseases,which led to the concept of the gut-lung axis.Gut-lung microbiota and metabolites are connected by a complex bidirectional axis via lymphatic and blood circulation,resulting in directly impact distant mucosal site by modifying mucosal barrier.However,data on this topic are scarce and the mechanisms by which the gut microbiota affects LC are poorly understood and remain to be studied in detail.Therefore,elucidating the relationship between gut microbiota and the pathogenesis of LC is of vital significance for early screening and treatment of LC.Therefore,the purpose of the study is to identify characteristic microbiota,metabolites and genes that play important roles in LC using 16S rRNA amplicon sequencing,metagenomics,metabolomics and transcriptomics,with the expectation predicting the correlation between gut microbiota,lung microbiota and pathogenesis of LC and providing new targets for the prevention,screening and treatment of LC.Methods:(1)Study design and sample collection:From 2021 to 2022,patients were recruited and the fecal,plasma,bronchoalveolar lavage fluid,lung tissue flushing solutions and tissue samples were collected in the Zibo Municipal Hospital,Shandong Province(No.20201102).All patients were categorized as lung cancer group(LC)and control group(C)according to the imaging and pathological diagnosis.(2)Bioinformatics analysis:① Differential analysis of the microbiota and metabolism in lungs.Data based on 16S rRNA amplicon sequencing,metagenomics and metabolomics were analysed to excavate the characteristics of the lung microbiota and metabolites in patients and verify potential biomarkers for diagnosis of lung cancer.② Metagenomics analysis of gut microbiota.Carrying out the sequencing of fecal samples(n=40)on Illumina,obtaining annotations of species and gene functions by DIAMOND and observing the taxonomic and functional differences of gut microbiota in two groups by Partial Least Squares Discriminant Analysis,Orthogonal Partial Least Squares Discriminant Analysis,Kruskal-Wallis sum-rank test and Networkx.③ Non-targeted metabolomics analysis of plasma.Metabolites in plasma samples(n=60)were detected by liquid chromatography-tandem mass spectrometry the information of metabolites were matched by Progenesis QI.Principal Component Analysis,Wilcoxon Signed Rank Test,Receiver Operator Characteristic curve and Spearman’s rank correlation coefficient were used to explore the changes of metabolomics profiles.④Transcriptomics analysis of lung tissues.Carrying out the sequencing of lung tissues(n=20)on Illumina and using the RSEM,DESeq2,KOBAS and Goatools to compare the differences of gene and miRNA expression in two groups.Results:(1)Changes in the microbiota and metabolism in lungs:Significant differences of lung microbiota reflected the changes of microbial beta diversity,distribution of microbial taxa,and the correlation networks of lung microbiota in lung cancer patients.Multiple oral bacteria were enriched in LC group,while potiential probiotics were enriched in controls.40 different metabolites,such as 9,10-DHOME,sphingosine,and cysteinyl-valine,were statistically significant between two groups.Moreover,the study found 3 metabolites and 9 species were significantly differences in LC group compared with the controls,which might be regarded as the potential clinical diagnostic markers associated with lung cancer.(2)Metagenomics analysis of gut microbiota:Microbiota associated with neurological diseases and the expression of genes and enzymes related to bile acid metabolism displayed significant differences between the two groups.Moreover,the gut microbiota of lung cancer patients may be influenced by the Enterobacter,Enterococcus,and Clostridium,which could potentially impact the disease through Microbial metabolism in diverse environments,ABC transporters and Biosynthesis of secondary metabolites.(3)Non-targeted metabolomics analysis of plasma:The results showed that lipid metabolism was disrupted in LC patients,with significant downregulation of multiple substances such as glycerophospholipids and phosphatidylcholine.Bile acid metabolism might exert significant influences on the pathogenesis of LC and 5-(hydroxymethyl)-2Furancarboxylic acid,2,4-Dihydroxy-2,5-dimethyl-3(2H)-furanone,PC(16:0/18:2(9Z,12Z)),GPCho(16:0/20:4)and MG(0:0/22:4(7Z,10Z,13Z,16Z)/0:0)might serve as the specific microbial signature for LC.(4)Correlation analysis of differential microbiota and metabolites:Prevotella,Allosalinactinospora lopnorensis,Akkermansia and Bacteroides might exert antagonistic effects with Blautia,Bifidobacterium mongoliense,and Chryseobacterium cucumeris,collectively influencing the progression of LC.(5)Transcriptomics analysis of lung tissues:Gene pathways associated with cell adhesion,transfer,and immunity,such as classical antibody-mediated complement activation,exhibited significant differences between the two groups.Analysis of target gene prediction of differential miRNA revealed that the epidermal growth factor receptor signaling pathway played a crucial role in tumor progression and immune evasion.Finally,10 miRNA-target gene pathways were identified by comparing the predicted target genes with the results of differential gene analysis,which might significantly influence tumor initiation and progression.Conclusion:Multiple omics analyses were performed to reveal changes in lung and gut microbiota,host metabolomics and gene expression profiles between LC patients and patients with nonlung cancer.The results suggested that the gut microbiota might mediate the secondary bile acid metabolism and regulate lung immunity through circulation,resulting in the influence on the occurrence and development of LC.The study explored the pathogenesis of LC in the interaction of the gut-lung axis from a new perspective and identified potential specific biomarkers for early diagnosis and screening of LC,which could provide new insights for targeted therapy of LC. |
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