The Microbial Analyses In Larynx And The Study Of Relationship Between The Microbiota And Laryngeal Squamous Cell Carcinoma

Laryngeal carcinoma is a common form of head and neck cancer, and more than95%of all laryngeal malignancies are squamous cell carcinomas. The main risks include cigarette smoking, alcohol drinking, and human papillomavirus. It is suggested that Helicobacter pylori (H. pylori) probably related to laryngeal carcinoma, and may be a risk factor to this type of cancer, but the exact mechanisms of carcinogenesis need further explained. Besides, it is still unknown if single species or a microorganic coordination are responsible for the pathogenesis. A current trend in the understanding of the human microbiome is a holistic view of the microbial community as the unit to develop and maintain microecology homeostasis and healthy barrier function. These microorganisms work together to act both synergistically and competitively. The disruption of this bio-ecological niche might give rise to the disease outcomes.In present research, three parts were applied to analyze the bacteria and its association with laryngeal squamous cell carcinoma (LSCC):to investigate the presence of H. pylori in the larynx, and to identify the relationship between H. pylori infection and LSCC; to analyze the expression of MSH2and MLH1in LSCC patients and explore whether their expression levels were altered by H. pylori infection; to inspect characteristics of microbial biology in laryngeal mucosa and investigate the abundant composition between LSCC patients and a control population.Part one, association between H. pylori infection and LSCC.The purpose of this part was to investigate the presence of H. pylori in the larynx, and to identify the relationship between H. pylori infection and LSCC. Eight one patients with LSCC and75controls (patients with vocal cord polyps and epiglottic cysts) were enrolled in a case- control study. Nested polymerase chain reaction (nPCR) techniques and sequence analyses were applied to detect H. pylori in the laryngeal mucosa. Enzyme linked immunosorbent assays (ELISA) were used to detect serum antibodies against H. pylori. The presence of H. pylori in the larynx was higher in patients with laryngeal cancer than in control subjects (71.6vs.25.3%, p=0.001). H. pylori infection was significantly higher in adjacent normal tissue samples compared with tumor tissues from patients with LSCC (p<0.001). After adjusting for confounding factors, regression analysis indicated that the microbe was a risk factor for laryngeal cancer (OR=8.12,95%CI [3.07,21.53], p<0.001). However, the result was not significant from ELISA investigation. Taken together, it is suggests that H. pylori is present in the mucosa of the larynx. The microorganism may be a risk factor for LSCC. The laryngeal mucosa thus provides a reservoir for the bacteria possibly, and is a likely staging place for its transmission to other areas.Part two, the altered expression of MSH2and MLH1induced by H. pylori in LSCC.The aim of this part was to investigate the expression of MSH2and MLH1genes expression in LSCC samples and analyze the relationship between MSH2and MLH1expression and overall survival rate in LSCC patients. We also wanted to evaluate whether their expression levels were altered by H. pylori infection. Using real time polymerase chain reaction (RT-PCR) and western blotting, we detected MSH2and MLH1expression in21laryngeal cancer tissue samples. We collected a retrospective cohort with180LSCC patients, and inspected MSH2and MLH1staining with tissue microarray (TMA) immunohistochemistry. Prognostic value of clinicopathological features was evaluated by statistic analysis. Laryngeal carcinoma cells were co-cultured with H. pylori. MSH2and MLHl were at a lower expression level than that of adjacent tissues in21LSCC patients (p<0.001). Patients with negative expression of MSH2and MLH1genes tended to have higher risk of death than patients with positive expression (MSH2:HR=4.38,95%CI:1.05-18.25; p=0.04; MLH1:HR=3.0,95%CI:1.76-5.14; p<0.001). H. pylori infection reduced the MSH2and MLHl expression levels of laryngeal carcinoma cell lines within co-culture condition (p<0.05). Taken together, it is suggested that the altered expression of MSH2and MLH1probably affect the overall survival of laryngeal carcinoma patients. Colonization of H. pylori in larynx might work as a potential role to impact the expression of MSH2and MLH1in laryngeal carcinoma patients.Part three, the composition of laryngeal microbiota and the distributional shift from commensal ecological structures to community of LSCC.The purpose of this part was to investigate the microbial biology of the larynx and to analyze the throat biodiversity in laryngeal carcinoma patients compared to a control population in a case-control study. We collected swab samples from40patients with LSCC and31controls, and also collected the matched tissue specimens.454Genome Sequencer FLX Titanium platform was applied to analyze the16S rRNA gene. The findings of high-quality sequence datasets revealed218genera from14phyla in the laryngeal mucosa. The predominant communities of phyla in the larynx mucosa were:Firmicutes (59.0%), Proteobacteria (10.8%), Bacteroidetes (10.4%), Fusobacteria (10.1%), Actinobacteria (10.0%)。The main community of phyla in the LSCC were:Firmicutes (52.5%), Fusobacteria (16.2%), Bacteroidetes (16.5%), Proteobacteria (11.0%), Actinobacteria (5.5%). The bacterial profiles from location of epiglottis and pharynx were different compared with community of laryngeal mucosa (p<0.001). The throat bacterial compositions were highly different in laryngeal carcinoma subjects compared with controls (p=0.006). Several bacterial communities might be associated with laryngeal carcinoma (p<0.001). In summary, this study reveals the microbiota profiles in larynx. There is a composition shift from commensal ecological structures to community of laryngeal cancer. The genera of Peptostreptococcus, Prevotella, Parvimonas, Streptococcus, Granulicatella and Helicobacter may be associated with laryngeal carcinoma. These microorganisms might coordinate to maintain a healthy environment in the throat, and the disorder of this dynamic homeostasis probably causes carcinogenesis in larynx.