Study of the Relationship between Microbiome and Colorectal Cancer Susceptibility Using 16SrRNA Sequencing

Abstract

A lot of previous studies have recently reported that the gut microbiota influences the development of colorectal cancer (CRC) in Western countries, but the role of the gut microbiota in Chinese population must be investigated fully. The goal of this study was to determine the role of the gut microbiome in the initiation and development of CRC. We collected fecal samples of 206 Chinese individuals: 59 with polyp (group P), 54 with adenoma (group A), 51 with colorectal cancer (group CC), and 42 healthy controls (group HC).16S ribosomal RNA (rRNA) was used to compare the microbiota community structures among healthy controls, patients with polyp, and those with adenoma or colorectal cancer. Our study proved that intestinal flora, as a specific indicator, showed significant differences in its diversity and composition. Sobs, Chao, and Ace indexes of group CC were significantly lower than those of the healthy control group (CC group: Sobs, Chao, and Ace indexes were 217.3 ± 69, 4265.1 ± 80.7, and 268.6 ± 78.1, respectively; HC group: Sobs, Chao, and Ace indexes were 228.8 ± 44.4, 272.9 ± 58.6, and 271.9 ± 57.2, respectively). When compared with the healthy individuals, the species richness and diversity of intestinal flora in patients with colorectal cancer were significantly reduced: PCA and PCoA both revealed that a significant separation in bacterial community composition between the CC group and HC group (with PCA using the first two principal component scores of PC1 14.73% and PC2 10.34% of the explained variance, respectively; PCoA : PC1 = 14%, PC2 = 9%, PC3 = 6%). Wilcox tests was used to analyze differences between the two groups, it reveals that Firmicutes (P=0.000356), Fusobacteria (P=0.000001), Proteobacteria (P=0.000796), Spirochaetes (P=0.013421), Synergistetes (P=0.005642) were phyla with significantly different distributions between cases and controls. The proportion of microorganism composition is varying at different stages of colon cancer development: Bacteroidetes (52.14%) and Firmicutes (35.88%) were enriched in the healthy individuals; on the phylum level, the abundance of Bacteroidetes (52.14%-53.92%-52.46%-47.06%) and Firmicutes (35.88%-29.73%-24.27%-25.36%) is decreasing with the development of health-polyp-adenomas-CRC, and the abundance of Proteobacteria (9.33%-12.31%-16.51%-22.37%) is increasing. PCA and PCOA analysis showed there was no significant (P < 0.05) difference in species similarity between precancerous and carcinogenic states. However, the composition of the microflora in patients with precancerous lesions (including patients with adenoma and polyp) was proved to have no significant disparity (P < 0.05). Our study provides insights into new angles to dig out potential biomarkers in diagnosis and treatment of colorectal cancer and to provide scientific advice for a healthy lifestyle for the sake of gut microbiota.

Figure 1

(a) Venn diagram of OUT and (b) dilution curve of the alpha diversity index. As is shown, the dilution curve of Sobs index with the curve is going to flatten out. (c) Principal component analysis (PCA) based on OTU abundance between groups CC and HC. x-axis, 1st principal component and Y-axis, 2nd principal component; 14.73% in brackets represents contributions of PC1 components to samples, 10.34% represents contributions of PC2 components to samples. A dot represents each sample, and different colors represent different groups (red: group CC and blue: group HC). (d) OUT rank curve.

Figure 2

Alpha diversity indices boxplot between group CC and HC. The abnormal value is shown as “o.”

Figure 3

Principal coordinates analysis (PCOA) between group HC and group CC. The figure shows the three-dimensional diagram of PCOA, in which each dot represents a sample, and each color represents a group: red for group CC and blue for group HC. PC1 is the principal coordinate component causing the largest difference in samples, with an explanatory value of 14%. PC2 and PC3 were next, with an explanatory value of 9% and 6%, respectively.

Figure 4

The taxonomic composition distribution in samples of the Phylumlevel. Relative abundance of bacterial phyla in microbiota in the healthy control group (HC) and relative abundance of bacterial phyla in microbiota in the CRC group (CC).

Figure 5

Proportions of main bacteria of the group CC and HC at the level of phylum (a, b) and genus (c, d). The pie chart of species proportion was obtained by calculating absolute abundance at the phylum and genus level, the major species are selected with a proportional advantage, and the percentage is accurate to 2-3 decimal places.

Figure 6

The taxonomic composition distribution in samples of the genus level. Relative abundance of bacterial genus in microbiota in the CRC group (CC) and relative abundance of bacterial genus in the healthy control group (HC).

Figure 7

Alpha diversity indices boxplot among four groups. Analysis of the Chao, Sobs, Ace richness index, Shannon index, and Simpson diversity index in A-CC-HC-P groups. (a) Boxplots of the Sobs richness index. (b) Boxplots of the Chao richness index. (c) Boxplots of the Ace richness index. (d) Boxplots of the Shannon diversity index. (e) Boxplots of Simpson diversity index.

Figure 8

Principal component analysis (PCA) based on OTU abundance. (a) Group HC and P, PC1 (16.63%) and PC2 (8.65%). (b) Group A and HC, PC1 (16.69%) and PC2 (8.41%).

Figure 9

Unweighted UniFrac principal component analysis (PCOA). The microbiota of healthy controls and individuals with polyps (a) as well as healthy controls and individuals with adenoma (b) were significantly different; no difference was found in microbiota composition of individuals with polyps and individuals with adenoma (c).

Figure 10

Unweighted UniFrac principal component analysis (PCOA) among groups. Both the 3D images indicate that there does not exist a distinct separation between group A, P (which is considered as precancerous diseases), and group CC.

Figure 11

Proportions of main bacteria of the (a) group A and (b) P at the level of phylum.

Figure 12

The trend chart based on the main microbial composition for each group at the phylum level. On the phylum level, the abundance of Bacteroidetes and Firmicutes is decreasing with the development of health-polyp-adenomas-CRC, and the abundance of Proteobacteria is increasing.