Exploration Of Distal Regulatory Mechanism Of "Lung And Large Intestine Being Interior Exteriorly Related" Based On Association Of "Intestinal Flora-circulating MiRNA-pulmonary And Intestinal Mucosal Immunity"

Objective: By analyzing the correlation between the abundance of intestinal flora and the expression level of circulating micro RNAs(miRNAs),we screened circulating miRNAs associated with intestinal flora,and further predicted the co-regulatory effects of circulating miRNAs on immune-related genes in the pulmonary and intestinal mucosal immunity,thus making a preliminary exploration of the " Lung and large intestine being interior exteriorly related ".Methods: Patients with intestinal flora imbalance,that is chronic constipation were recruited for follow-up study.Stool samples and whole blood samples were collected on day 0(the first time point),day 28(the second time point)and day 42(the third time point)after enrollment.The abundance of intestinal flora in stool samples and circulating miRNA expression profile in plasma samples were detected by high-throughput sequencing technology.The correlation between the abundance of intestinal flora and circulating miRNA expression profile at each time point was analyzed by R software,and the miRNAs associated with intestinal flora with good repeatability were screened out.Target genes of miRNAs associated with intestinal flora were obtained by Target Scan and mi Randa target gene prediction software,and the roles of target genes in pulmonary and intestinal mucosal immunity were further analyzed by GO functional enrichment analysis and KEGG pathway enrichment analysis platformResult:(1)A total of 24 qualified patients with chronic constipation were recruited,including 3 males and 21 females;The average age was 20.38 years old,and all of them had bachelor’s degree or above;A total of 70 stool samples and 72 whole blood samples were collected and tested.(2)A total of 433 species of intestinal flora and 1727 species of circulating miRNAs were detected.The results of association analysis showed that there were 378 pairs of miRNAs associated with intestinal flora with strong association and good repeatability.Among them,368 pairs of miRNAs associated with intestinal flora were correlated at two time points,183 pairs were positively correlated,and 185 pairs were negatively correlated.The 10 pairs of miRNAs associated with intestinal flora were correlated at three time points,with 5 pairs positively correlated and 5 pairs negatively correlated.A total of 78 intestinal flora and 267 circulating miRNAs were involved.(3)According to the target gene prediction analysis,267 intestinal flora associated miRNAs could target 817 pulmonary mucosal immune genes and 346 intestinal mucosal immune genes,among which 113 were pulmonary and intestinal mucosal immune co-expressed genes.These genes are mainly divided into interleukins,interferons,tumor necrosis factors,colony-stimulating factors,growth factors,chemotactic factors,human leukocyte antigen and others.GO function enrichment results showed that the functions of these genes mainly focused on the production of cytokines/leukocyte surface antigens and their mediated signaling pathways,response to xenobiotic stimulus,inflammatory response and immune response,and other biological processes;The molecules/proteins expressed were mainly distributed in cell components such as tight junctions,mitochondria,ribosomes and cell surfaces;The expressed molecules mainly have molecular functions such as cytokine activity and protein binding.KEGG pathway enrichment analysis results showed that: These genes were mainly enriched in colorectal cancer,non-small cell lung cancer,epstein-barr virus infection infection,inflammatory bowel disease,influenza A,tuberculosis,measles,Fox O signaling pathway,Th17 cell differentiation and apoptosis.Conclusion: These results indicate that there may be a large number of circulating miRNAs associated with intestinal flora in human plasma,and the expression of these circulating miRNAs can change with the abundance of intestinal flora,and then regulate the expression of immune-related genes in lung and intestinal mucosa,which plays an important role in the mechanism of "lung and large intestine being interior exteriorly related".This study has completed the preliminary exploration of the molecular mechanism of "lung and large intestine being interior exteriorly related" from the perspective of "intestinal flora-circulating miRNA-pulmonary and intestinal mucosal immunity",but further experimental confirmation is still needed.