| Objective: Colorectal cancer is one of the common gastrointestinal malignancy. So far, it remains unclear about the etiology and pathogenesis of colorectal cancer. Previous studies suggest that a variety of factors involved in the development of colorectal cancer, including diet, obesity, exercise and genetic factors such as the individual. Recent studies have found that changes in the intestinal microflora and metabolic patterns may play an important role in the development of colorectal cancer. In addition, the treatment of colorectal cancer is relatively mature, including surgical treatment, combined with chemotherapy, radiotherapy, immunotherapy, Chinese medicine and other means. Up to 90% of patients can be cured by surgery if the disease is detected at an early stage, but unfortunately it is often diagnosed only at an advanced stage and the prognosis is therefore poor. The aim of this study is to explore characteristic changes of intestinal microbiota in colorectal cancer patients based on the characteristics of China’s population, to find specific carcinogenic bacterium like Helicobacter pylori bacteria in the gut and investigate the correlation between the specific carcinogenic bacterium and colorectal cancer prognosis.Methods:(1) Clinical samples were collected. Fresh colorectal cancer and adjacent normal mucosal samples(35 cases), inflammatory bowel disease(IBD, including Crohn’s disease and ulcerative colitis, 20 cases) mucosal samples and samples of colorectal adenomas(15 cases) from the digestive Endoscopy Center of Shanghai Jiaotong University Affiliated six People’s Hospital from September 2011 to September 2012, confirmed by histopathology. All colorectal cancer patients need further surgery. Stool samples of colorectal cancer patients were collected a week after endoscopy(intestinal microbiota returned to normal during this time); Oral probiotics(Bifico powder) and placebo for one week were performed in 22 colorectal cancer patients before surgery. Intraoperative colorectal mucosal samples were collected. In addition, normal intestinal mucosal and stool samples of 15 healthy volunteers were provided by community residents in Zhabei District, Shanghai. All samples are in line with the appropriate inclusion and exclusion criteria, and the project was approved by the institutional review board of the Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University, China. Written informed consent was obtained from all patients and tissue sample donors, and anonymity was maintained by tracing patients through their clinical history number.(2) The detection of intestinal microbiota structural changes in patients with colorectal cancer. Genomic DNA were extracted utilizing QIAamp DNA specimens kit and were examed by 1% agarose gel electrophoresis, then the genomic DNA content for each samples were identified; Extracted genomic DNA was amplified by PCR using ABI Gene Amp and examed with a 2% agarose gel electrophoresis, followed by fluorescence quantitative; 16 S r RNA V3 variable region were pyrosequenced using Roche GS FLX 454 sequencer for PCR amplification products, After that, the sequences were aligned using SILVA database and the analysis of Good’s coverage, Diversity index, PCA, LEf Se were also adopted in order to explore the diversity and the structure changes of the intestinal microbiota.(3) A large sample validation of Fn and Pf bacteria. Paraffin-embedded CRC specimens and adjacent matched non-tumour tissue obtained from 283 patients who underwent surgical resections from January 2007 to December 2011 were analysed by quantitative reverse transcription PCR(q RT-PCR) and paraffin-embedded CRC specimens and adjacent matched non-tumour tissue from 95 independent patients who underwent surgical resections were analysed by fluorescence in situ hybridisation(FISH) choosed from Tenth People’s Hospital Affiliated to Tongji University. Among them, 144 cases of male patients, female 139 cases, aged 40-80 years, mean age 67 years.(4) Correlation analysis were performed between Fn and Pf expression levels and prognosis of colorectal cancer. We use 50 th percentile of mucosal cancer: paraneoplastic of normal mucosa to dichotomise CRC cases where low Fn expression was classified as the lower 50 th percentile and high Fn expression was classified as the upper 50 th percentile. The same is Pf, Kaplan-Meier method and Log Rank test were used for survival analysis. Univariate and multivariate analysis of colorectal cancer survival were performed using Cox proportional hazard model.(5) The effect of probiotic intervention on colorectal intestinal microbiota structure. Genomic DNA was extracted using QIAamp DNA Mini Kit on these probiotic intervention specimens according to the manufacturer’s instructions. The subsequent determination of the 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 explore the feasibility of probiotic intervention on colorectal cancer intestinal microbiota.Results:(1) Pyrosequencing results showed that the intestinal microbial composition differed significantly between patients with colorectal cancer and healthy control group. At the phylum level, The abundance of Firmicutes and Fusobacterium were elevated whereas the abundance of Proteobacteria was reduced in colorectal cancer compared with normal mucosa. Although the abundance of Bacteroides and Actinobacteria were higher in colorectal cancer, there was no significantly statistical difference. At the genus level, Lactococcus(50.85% vs. 25.35%, P=0.007), Fusobacterium(10.08% vs. 0.01%, P=0.032), Escherichia-Shigella(2.92% vs. 0.22%, P=0.004) exhibit- ted a relatively higher abundance in CRC rats compared to healthy control. However, the abundance of Flavobacterium, Pedobacter, Sphingo bacterium, Caulobacter, Brevundimonas, Sphingomonas, Sphingomonas, Acidovorax, Buttiauxella, Rahnella, Acinetobacter, Psychrobacter, Pseudomonas, Steno- trophomonas and Psychrobacter was significantly reduced. Using discrimi- nant analysis coupled with effect size model(LEf Se), we find that Fusobac- terium, Prevotella and Bacteroides of patients with colorectal cancer constitute the “core microbiota” of intestinal mucosa, while Pseudomonas and Flavobacterium constitute the “core microbiota” of healthy population. The stool sample had a completely different microbiota structures compared to mucosa, The abundance of Firmicutes and Bacteroidetes was significantly reduced, while the abundance of Proteobacteria and Actinobacteria was significantly increased. We also compared microbial community structure among colorectal cancer tissues, matched adjacent tissues and stool samples. We found that there were significantly different between the tree groups. Bacteroidetes had the highest abundance in paraneoplastic mucosa, Proteobacteria had the highest abundace in fecal samples, while Fusobacteria had the highest abundance in CRC microenvironment.(2) By performing multiple sequence alignments using Fusobacterium and Pseudomonas along with 16 S r RNA sequences from a reference set, Fusobacterium nucleatum(Fn) and Pseudomonas fragi(Pf) were screened for further study. The result of q RT-PCR showed that Fn was enriched in colorectal mucosal surface, while Pf is significantly lower, which was consistent with our previous results. FISH results confirmed that part of the Fn can invade the lamina propria layer, suggesting that Fn may be involved in the development of colorectal cancer.(3) Univariate and multivariate analysis of prognostic Fn overexpression correlated with significantly reduced patient survival with colorectal cancer and was independent prognostic factors(HR=1.71,P=0.039). Although Pf expression level had no obvious correlation with prognosis of CRC patients, we stratified Fn expression level by Pf expression to explore the correlation to prognosis of CRC patients, we found a more significant correlation between Fn and prognosis. Therefor Fn and Pf can serve as a valuable molecular markers associated with prediction of the prognosis of patients after surgical removal of the tumor.(4) Probiotic intervention study showed that oral administration of probiotics can significantly improve the structure of the intestinal microbiota in patients with colorectal cancer, and reduce the abundance of Fn in intestinal mucosa. This result indicated that oral administration of probiotics can be use as a preventive and treatment tactics for patients with colorectal cancer.Conclusion:The microbial community structure in colorectal cancer patients had a characteristic changes, mainly in the change of the “core microbiota” including Fn and Pf, which may be involved in the development and progression of colorectal cancer; at the same time, Fn and Pf expression in tumor microenviroment are related with prognosis in patients with colorectal cancer. High Fn companied high Pf expression indicates a poor prognosis; In addition, probiotics intervention can significantly change the structure of the intestinal microbiota of patients with colorectal cancer and reduce the abundance of Fn. Accordingly, It is suggested that Fn and Pf should be considered as a potential biomarker as colorectal cancer risk screening, treatment strategies and prognosis of a potential target. |
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