| BACKGROUND Breast cancer is the most common malignant tumor in women in the world.The incidence rate is 7% to 10% of all malignant tumors.About1.4 million people were diagnosed with breast cancer,and about 500 thousand people die from this disease each year,which is a serious threat to women's health.The incidence of breast cancer may be related to endocrine,reproductive factors,diet,radiation and genetic factors,but the reasons are not fully understood.Intestinal microbes are influenced by genetic factors,lifestyle and environment,involved in the basic physiological activities of human body.Intestinal microbes are involved in maintaining the structure and physiological function of intestine,inhibiting the colonization of pathogenic microorganism,metabolism and immunity of human body.It maintain the health of human body.The occurrence and development of human diseases are closely related to the structure and function of intestinal microbes.A large number of studies have shown that human intestinal microbes were closely associated with obesity,diabetes,ulcerative colitis,Crohn's disease,neonatal necrotizing enterocolitis,cancer and other diseases.There is growing evidence that gut microbes are associated with breast cancer.The mechanism may through estrogen metabolism,diet,immune regulation,antibiotics,alcohol,inflammation and other factors.At present,the study of intestinal microflora of breast cancer is limited to the community structure of intestinal microflora,and lack of the study of function of intestinal microflora in breast cancer.The understanding of intestinal microbes depends mainly on the research technique.The traditional Microbiology and microbial genomics are based on microbial culture,but more than90% of the intestinal microbes cannot be cultured,cultivation method used to study the diversity of gut microbes will cause serious deviation.Although the 16 Sr RNA gene sequencing analysis can be used for species classification and abundance analysis,and used to study the structure of intestinal microorganisms,but it can not be systematically used to study the function of the intestinal microbes.With the rapid development of genomics,the study of intestinal microorganisms has entered into the era of metagenomic research,with the high-throughput sequencing technology,we can more systematically and comprehensively study the structure and function of intestinal microflora in patients with breast cancer.OBJECTIVE In this study,next-generation genome sequencing tech-nology was used for the first time to systematically analyze the changes of structure and function of intestinal microflora in postmenopausal women with breast cancer.To investigate whether there is an imbalance of intestinal microflora in breast cancer patients,and to identify the key microbes associated with breast cancer patients.In addition,to investigate the correlation between the changes of intestinal microbial function and breast cancer.To reveal the role of intestinal microflora in the development of breast cancer.This study provide the basis for further study of the pathogenesis of breast cancer,and is helpful to predict and evaluate the risk of breast cancer,and to develop new approach for diagnosis and treatment of breast cancer.METHODS Fecal samples were collected from 25 Premenopausal healthy volunteers,18 Premenopausal breast cancer patients,46 postmenopausal healthy volunteers and 44 postmenopausal breast cancer patients.The patients with pathological diagnosis of breast cancer,no diarrhea,diabetes,ulcerative colitis,Krohn's disease and other infectious diseases.Breast cancer patients and healthy control volunteers did not take antibiotics,steroid hormones and Chinese herbal medicine(including oral,intramuscular or intravenous injection),not taking probiotics or probiotics such as yogurt,unused cyclophosphamide,anthracycline and taxane chemotherapy drug in three months before the fecal samples collection.Genomic DNA was extracted and sequenced on Illumina Hi Seq X10.Metagenomics reads were used for species taxonomic profiling using BWA,and assignments to functional modules,orthologs,pathways through alignment to Kyoto Encyclopedia of Genes and Genomes(KEGG)also using BWA.RESULT1.In the overall analysis of the structure of intestinal flora in the four groups,in the phylum level,there were significant differences of Proteobacteria in the four groups.In the genus level,there were significant differences of Acidaminococcus,Acinetobacter,Actinomyces,Citrobacter,Epulopiscium,Erwinia,Escherichia,Klebsiell,Peptoniphilus,Proteus,Providencia,Rothia,Salmonella,Sodalis,Variovorax,Vibrio,Yersinia in the four groups.2.In the overall analysis of functional metabolism in the four groups,there were significant differences in the functional metabolism of intestinal flora between the four groups,including amino acid transport,vitamin transport,toxic products transport,antioxidant metabolism,replication and signal transduction related to bacteria.3.In the comparative analysis on the structure of intestinal flora between breast cancer patients and healthy controls,at phylum level,there was no significant difference of intestinal flora between premenopausal breast cancer patients and premenopausal healthy controls.Compared with postmenopausal healthy controls,the abundance of Fusobacteria,Proteobacteria and Synergistetes was significantly increased in postmenopausal breast cancer patients.At the genus level,there was no significant difference of intestinal microflora between premenopausal breast cancer patients and premenopausal healthy controls.Compared with postmenopausal healthy controls,the abundance of Escherichia,Shigella,Proteus,Vibrio,Acinetobacter,Providencia,Salmonella,Sodalis,Citrobacter,Thiomicrospira,Variovorax,Peptoniphilus,Pseudomonas,Erwinia,Yersinia,Actinobacillus and Bulleidi was significantly increased in postmenopausal breast cancer patients,the abundance of Actinomyces was significantly reduced in postmenopausal breast cancer patients.4.In the comparative analysis on functional metabolism of intestinal flora between breast cancer patients and healthy controls,compared with premenopausal healthy controls,the abundance of bacteria related to the metabolism of amino acids was significantly increased,and the abundance of bacterial related to the bacterial replication was significantly reduced in premenopausal breast cancer patients.Compared with postmenopausal healthy controls,the abundance of bacteria related to Vitamin transport,transport and metabolism of toxic products,signal transduction,replication,recombination and repair was significantly increased in postmenopausal breast cancer patients.5.In the analysis of the correlation between the significantly different bacteria of breast cancer patients and healthy controls and clinical phenotypes,the altered bacteria genera(Pseudomon)of postmenopausal healthy controls and postmenopausal breast cancer patientswas positively correlated with CD3+CD4+/CD3+CD8+(Th/Ts).The altered bacteria genera of postmenopausal healthy controls and postmenopausal breast cancer patients was not associated with humoral immunity and estradiol(E2).6.In the analysis of the correlation between the altered metabolic pathways(map)of breast cancer patients and healthy controls and clinical phenotypes,the altered metabolic pathway(map)of postmenopausal breast cancer and postmenopausal healthy controls was not associated with the cellular immunity and estradiol(E2),and there was a significant correlation between the metabolic pathway(map)and humoral immunity.7.In the analysis of the correlation between the altered KEGG module of breast cancer patients and healthy controls and clinical phenotypes,the altered module of premenopausal breast cancer and premenopausal healthy controls was significantly associated with cellular immunity and humoral immunity,and was not associated with estradiol(E2).The altered module of postmenopausal breast cancer patients and postmenopausal healthy controls was significantly associated with humoral immunity,and not associated with cellular immunity and estradiol(E2).CONCLUSION The community structure and functional structure of intestinal microflora in breast cancer patients were significantly altered,and the intestinal flora of patients with breast cancer was related to humoral immunity,cellular immunity.In this study,we systematically analyzed the community structure and function of intestinal microflora in postmenopausal women with breast cancer.The role of intestinal flora in the occurrence and development of breast cancer was revealed.The results suggest that the changes in the structure and function of the intestinal flora can be used as a biological indicator for predicting and evaluating the risk of breast cancer.It also may provide a theoretical basis for clinical diagnosis of breast cancer through intestinal flora. |
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