The Correlative Research Of The Effect Of Fusobacterium Nucleatum On Colorectal Carcinogenesis

Objective:Nowadays, colorectal cancer(CRC) has become one of the most common malignant tumors. However, the mechanism is not yet clear. Recent reports have suggested that multiple factors such as host genetics, environment and diet can promote the progression of healthy mucosa towards sporadic colorectal carcinoma. Especially, accumulating evidence has associated intestinal bacteria with disease initiation and progression. Recent studies have reported a high abundance of Fusobacterium in CRC subjects compared to normal subjects. Our studies also confirmed previous reports on Fusobacterium and CRC tumor and matching normal tissues by pyrosequencing. In order to observe the effect of fusobacterium on the incidence of colorectal carcinoma and investigate the potential relationship between changes of fusobacterium abundance and colon carcinogenesis, an azoxymethane(AOM) induced C57 rat CRC model mimicking human colorectal carcinogenesis was established.Methods:(1) The Fn bacterium and colon cancer cell lines HCT116 cells were co-culture. The ability of Fusobacterium bacterium adhesion and invasion of colon carcinoma epithelial cell was measured. Screening of differentially expressed genes from HCT116 cell infected fusobacterium by gene chip,(2) The establishment of in situ animal model of colorectal cancer. 8 to 10-wk-old C57 mice were divided into groups randomly, and were acclimatized to the environment for 1wk. After that, animals were intraperitoneally injected with AOM or saline once for a week. A week later, the mice were fed with bacteria or saline once a day for five consecutive days. And then the mice were subjected to the same cycle. From the beginning, at 81 th day of the protocol, mice were sacrificed in order to examine the formation of colon tumors. After that, tumors were sectioned and stained with hematoxylin and eosin for histological examination. The levels of serum IL-6, IL-8, COX-2, TNF-α and so on were measured using corresponding ELISA kits. To explore the underlying mechanisms at the molecular level, the analysis of gene levels in tissue and the expression of upstream regulatory factors were determined using Western blotting and RT-PCR.(3) The determination of structure changes of the intestinal microflora in CRC animal model. Nucleic acids were extracted from each intestinal stool samples using the QIAamp DNA stool Mini kit. Integrity and size of DNA was determined by 1% agarose gel electrophoresis. The process of PCR amplification for genomic DNA was conducted on an ABI Gene Amp cycler, and amplified products from stool samples were verified by gel electrophoresis using 5μl of the PCR reaction mixture in a 2% agarose gel. The products from different samples were mixed for pyrosequencing using the Roche GS FLX 454 Sequencer according to the instructions. After that, the sequences were aligned using SILVA database and the analysis of Good’s coverage, Shannon index, PCA, LEf Se were also adopted in order to determine the diversity and find the structure changes of the intestinal microflora.(4) The determination of the structure changes of gut microbiota and the exploration about the relationship between the structure changes and the incidence of tumors under fusobacterium. After the tissue samples were collected, genomic DNA was extracted using the QIAamp DNA Mini Kit according to the manufacturer’s instructions. Afterwards, the determination of genomic DNA, PCR amplification, fluorescence quantitation, pyrosequencing of 16 S r RNA V3 region and the bioinformatic analysis were performed as described above in order to determine the key bacterial phylotypes that might play important roles in the development of CRC.Results:(1) The Fusobacterium bacterium can adhere to and invade colon epithelial cell. Screening of differentially expressed genes from HCT116 cell infected fusobacterium by gene chip, in which PCNA, BIRC3 and NF-κB genes expression was significantly upregulation.(2) The CRC in situ animal model was successfully established, which mimicked the development of human CRC carcinogenesis. The number of colonic tumors was significantly higher in the Fn group than in the SD group. The Fn group also have increased levels of serum IL-6, IL-8, COX-2, TNF-α. Fn induced upregulation of PCNA, BIRC3 and NF-κB using Western blotting and RT-PCR.(3) Our findings indicate that the microbial composition of the intestinal lumen differs significantly between control and Fusobacterium tumor groups. The abundance of Firmicutes was elevated whereas the abundance of Bacteroidetes was reduced in the lumen of CRC mice. Fusobacteria was not detected in any of the healthy mice. There was no significant difference in observed groups. However, the abundance of Proteobacteria was higher in CRC mice. At the genus level, we also demonstrate a significant reduction of butyrate-producing bacteria such as Roseburia and Eubacterium in the gut microbiota of CRC mice. Furthermore, a decrease in probiotic species such as Ruminococcus was likewise observed in the tumor group, and significant increase in Fusobacterium was also observed in the tumor group. Bacteroides exhibited a relatively higher abundance in CRC mice compared to controls(2.12% vs. 0.31%, p<0.001). Meanwhile, Lactobacilluswere found to be significantly more abundant in healthy mice than CRC mice(60.50% vs. 45.88%, p<0.001).Conclusion:Fusobacterium bacterium can adhere to and invade colon epithelial cell, promote colon cell proliferation, inhibition of apoptosis, mediate the inflammatory process. Using in situ CRC animal models, we demonstrated that Fn could promote the formation of colonic tumors through inflammation, increased cell progression. Fn can change microbial community structure, increasing the abundance of pathogens, suggesting that this species might play an important role in the development of colorectal cancer.