Study On The Inflammation Induced By Fusobacteria Necleatum In Intestinal Epithelial Cell

Fusobacterium nucleatum(F. nucleatum) was a close association with inflammatory bowel disease(IBD) and colorectal cancer(CRC), which studies were published in Genome Research in 2012. Katrina Ray, the chief editor of Nature Reviews Gastroenterology&Hepatology, claimed that both of the researches have made tentative steps towards establishing an infectious cause of CRC by finding the periodontal pathogen F. nucleatum in colon cancer tissue. In addition, these studies were also considered to be one of the ten most important medical breakthroughs by Time Magazine in 2012. In recent years, accumulating microbiologists and clinicians pay great attention and interest on the correlation between F. nucleatum and gastrointestinal diseases.It is well known that IBD is an important risk factor for CRC. The chronic persistent inflammation of IBD stimulates cells proliferation and oxidative stress, ultimately leading to dysplasia and carcinogenesis. However, etiology and pathogenesis of IBD has not yet been fully elucidated. The latest finding of F. nucleatum provides us a new clue to study IBD and CRC. Thus, with further study on the mechanism of chronic persistent infection caused by F. nucleatum, it has an important theoretical significance to reveal the pathogenic mechanism and develop new and effective treatments to prevent and cure colorectal cancer.Chip technology has become an important research platform in gene expression and regulation with its advantages, including high throughput, the high automation degree, the fast detection speed and others. In this study, we apply this technology to screen inflammation and cancer-related genes caused by F. nucleatum from colorectal cancer tissues, and analyze the role of inflammatory signaling and cancer signaling pathways in the development of colorectal cancer. Furthermore, we developed the cell model of F. nucleatum infecting intestinal epithelial cell, and detected inflammatory response, autophagy and aptosis induced by F. nucleatum at different time points with several technologies, including Western blot, RT-PCR, ELISA and others. This paper opens a new avenue of research model to modulate IBD and colorectal cancer in F. nucleatum infection, and provides a theoretical basis for new treatment of colorectal cancer induced inflammation.Results1. Quantitative real-time polymerase chain reaction(q PCR) experiment showed that the colonization of F. nucleatum is much higher in disease tissues versus matched normal tissues in 113 cases of CRC patients(p=0.012), but it is not relevant to the age, gender, tumor location, staging, metastasis.2. A total of 1074 m RNAs and 49 mi RNAs was differently expressed in colon tumor tissues with or without F. nucleatum infection, while these m RNAs and mi RNAs might mainly related to inflammation, autophagy and cancer signal pathways.3. The protein and m RNA expression of IL-1β, IL-8 and TNF-α were detected at different time by ELISA and Real-Time PCR in the cell modle of F. nucleatum infection, respectively. Our results showed that the three inflammatory cytokines were significantly higher than the control group at the transcriptional level or protein level, while peaked at 6h.4. F. nucleatum could disrupt autophagy in intestinal epithelial cells. Our results showed that it promoted LC3 B accumulation, down-regulated ATG16L1 protein expression, and induced the expression of proinflammatory cytokines IL-8. Moreover, with autophagy inhibitor(Baf. A1) pretreatment, there were no significant difference in the expression of LC3 B and IL-8 with F. nucleatum infection.5. F. nucleatum could induce apoptosis in intestinal epithelial cells. Our results showed that it down-regulated AKT and p-AKT proteins expression and the GADD34 and GADD153 were significantly higher than the control group at the transcriptional level.Conclusions1. The content of F. nucleatum in colon cancer was significantly higher than the next adjacent tissues, and the differentially expressed genes were screened with m RNAs and mi RNAs microarray assay. These results indicated that F. nucleatum could regulated inflammation, autophagy, cancer and other signaling pathways, and it could involved in the occurrence and development of colorectal cancer.2. F. nucleatum could induce the expression of pro-inflammatory cytokines IL-8, IL-1β, TNF-α in intestinal epithelial cells Caco-2, suggesting that these pro-inflammatory cytokines may play an important role on inducing IBD.3. F. nucleatum infection could hinder the mature of autolysosomes with inhibiting Atg16L1, weak the autophagic clearance of the bacteria, and enhance the inflammatory response, leading to persistent infection and IBD.4. F. nucleatum can induce Caco-2 intestinal epithelial cell apoptosis, and the mechanism may be associated with reduced phosphorylation of AKT and elevated transcription level of GADD34 and GADD153 genes.