| Colorectal cancer (CRC) is one of the cancers with highest morbidity and mortality in the world. In the recent decades, both epidemiological surveys and molecular cell biology studies have indicated that improper diet is the most significant risk factor of CRC. Incidence of up to 80% of CRC has been attributed to dietary factors in western populations. The human gut microbiota, which anaerobically ferments dietary components not completely digested and absorbed in the upper gastrointestinal tract, is thus suggested to have significant roles in influencing CRC risk through their interactions with host metabolism and immunity. Development of diseases such as obesity, metabolic syndromes and inflammatory bowel diseases has been tightly associated with gut dysbiosis by many researchers. The present study aims at investigating the structural shifts of gut microbiota during the occurrence and development of CRC through both animal trials and human cohort study, and helping with understanding the roles of gut microorganisms in maintaining host health. Firstly, we have induced precancerous lesions aberrant crypt foci (ACF) in rats'colon using twice of injections of chemical carcinogen 1,2-dimethylhydrazine (DMH), and monitored the structural shifts of rat gut microbiota during the formation of ACF. Representing the gut bacterial phylogenetic diversity, 16S rRNA genes were profiled by various molecular tools including 454 pyrosequencing and two kinds of DNA fingerprinting methods: terminal restriction fragment length polymorphism (T-RFLP), and fluorophore-ribosomal DNA restriction typing (f-DRT), a new DNA fingerprinting technology we have developed in the present study. Both principal component analysis (PCA) and principal coordinate analysis (PCoA) based on UniFrac metric showed that, the gut microbiota structure of DMH treated rats were similar with that of control rats at an early stage (one week after the second injection), but segregated from that of controls when ACF appeared in their colons (37.7±2.6/whole colon) whereas no ACF in the colons of control rats. By the partial least square-discriminant analysis (PLS-DA) model established with pyrosequencing data, we identified bacterial groups which acted as key variables for the discrimination of DMH-treated rats from controls, including two bacterial groups closely related to Ruminococcus obeum (with 99.3% similarity) and Allobaculum stercoricanis (with 93.7% similarity), which were significantly more abundant in the gut microbiota of DMH-treated rats with ACF in their colon (p<0.05). These findings were consistent with a previous study of our lab on the same samples using denaturing gradient gel electrophoresis (DGGE). The significant changes of the host's gut microbial structure might be caused by the formation of colonic precancerous lesions, and then affect the early progress of colon cancer. Our results also showed that dynamic analysis of gut microbiota may become a noninvasive strategy for monitoring host health changes induced by carcinogen exposure.The second part of the present study is a cohort study including 46 CRC patients and 56 healthy volunteers. Fecal bacterial diversity in all volunteers was profiled by 454 pyrosequencing of the V3 region of 16S rRNA gene and multivariate statistics. We have observed significant differences of the gut microbiota structure between CRC patients and healthy population by PCA and PCoA based weighted UniFrac metric. Making up the significant differences, the amount of Bacteroidetes was significantly lower in the gut of CRC patients than healthy volunteers, while the amount of Proteobacteria was significantly higher. Redundancy analysis (RDA) was used to identify bacterial groups contributing significantly to the structural difference. Among these bacterial groups, some were potential opportunistic pathogens, which belonged to the genera Enterococcus, Escherichia/Shigella, Klebsiella, Streptococcus and Peptostreptococcus. These bacterial groups were significantly more abundant in the gut microbiota of CRC patients. Some other bacterial groups belonging to the butyrate producing genus Roseburia and other butyrate producing bacteria of the family Lachnospiraceae were less abundant in the gut microbiota of CRC patients. Four bacterial groups belonged to the genus Bacteroides and showed different enrichments between healthy volunteers and CRC patients. One closely related to Bacteroides fragilis (with 98.1% similarity) was enriched in the gut microbiota of CRC patients, while two other groups related to Bacteroides vulgatus (with 100.0% and 98.7% similarity respectively) and one related to Bacteroides uniformis (with 97.5% similarity) were enriched in that of healthy volunteers. Real-time quantitative PCR further validated the significant reduction of butyrate producing bacteria in the gut microbiota of CRC patients by measuring the copy numbers of butyryl-CoA CoA transferase gene (Mann-Whitney test, p<0.01), which is involved in the butyrate producing pathway of most butyrate producing bacteria in human gut. This part of work indicates that reduction of butyrate producers and increase of opportunistic pathogens may constitute a major structural imbalance of gut microbiota in CRC patients.In conclusion, we have indicated that, the structural shifts of gut microbiota may be tightly associated with the whole process of CRC. Dynamic monitoring of the structural segregation of gut microbiota away from the healthy majority may become a useful tool for pinpointing individuals whose health status indicates high CRC risk. The features of structural imbalance of gut microbiota in CRC patients revealed in this study may provide insights for in-depth analysis of host–microbe interactions and determination of their roles in cancer development. |
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