Enterocloster bolteae drives hyperglycemia-associated colorectal cancer cell proliferation via Rac/Rho-MSH6-Elongin C signaling pathway

Figure 1 Common and unique information of genes among different samples (groups). A: Degree distribution analysis of the gene catalog. The left vertical axis (frequency) represents the number of genes in the gene catalog; the right vertical axis (percentage, %) represents the percentage of genes in the gene catalog; the horizontal axis represents the degree of genes in the gene catalog; B: Heatmap of unigene annotation counts. The horizontal axis represents sample names; the vertical axis represents species information; different colors represent different numbers of unigenes; C: Horizontal relative abundance clustering heatmap. The horizontal axis represents sample information; the vertical axis represents species information. The clustering tree on the left is a species clustering tree. The values in the central heatmap correspond to Z-scores obtained by standardizing the relative abundance of each species per row. In this Z-score normalized heatmap, positive values (typically warmer colors such as red) indicate that the abundance/expression level of a given gene/species in a particular sample is higher than its mean level across all samples; higher magnitudes (e.g., +2 and +3) reflect greater deviation above the average. Negative values (typically cooler colors such as blue) indicate that abundance/expression levels below the overall mean; smaller values (e.g., -2 and -3) eflect greater deviation below the average. Values close to zero (often white) indicate that abundance/expression levels near the mean across all samples; D-F: Venn diagrams showing shared and unique genes across individual samples. Each circle represents one sample, with colors indicating group affiliation: Patients with diabetes mellitus (n = 8), patients with diabetes mellitus and colorectal cancer (n = 11), patients with colorectal cancer without diabetes (colorectal cancer, n = 10), and healthy controls (control, n = 11). Numbers in the overlapping regions denote the genes shared among samples, whereas numbers in the non-overlapping regions represent the unique genes identified in each sample; G: Box plot comparing gene counts between groups. The horizontal axis represents each group; the vertical axis represents the number of genes. Statistical differences in the number of genes among the four groups were evaluated using the Kruskal-Wallis H test. DM: Diabetes mellitus; CRC: Colorectal cancer; L.CRC: Left-sided colorectal cancer; R.CRC: Right-sided colorectal cancer.

Figure 2 Dimensionality reduction analysis based on species abundance. Each point in the figure represents a sample, and the distance between points represents the degree of difference. Samples from the same group are color-coded accordingly. The results show significant differences among the groups. A: Non-metric multidimensional scaling (NMDS) plot at the class level. A stress value less than 0.2 indicates that the NMDS analysis has reasonable reliability; B: Principal component analysis plot at the class level. The horizontal axis represents the first principal component, and the vertical axis represents the second principal component; percentages indicate the contribution of each component to sample variance; C: Principal coordinate analysis plot based on Bray-Curtis distance at the class level; D: NMDS plot at the species level. A stress value less than 0.2 indicates that the NMDS analysis has reasonable reliability; E: Principal component analysis plot at the species level. The horizontal axis represents the first principal component, and the vertical axis represents the second principal component; percentages indicate the contribution of each component to sample variance; F: Principal coordinate analysis plot based on Bray-Curtis distance at the species level; G: Bray-Curtis distance clustering tree (left) and relative abundance distribution of functions at the class level for each sample (right); H: Relative abundance distribution at the species level for each sample. DM: Diabetes mellitus; CRC: Colorectal cancer; NMDS: Non-metric multidimensional scaling; PCA: Principal component analysis; PCoA: Principal coordinate analysis.

Figure 3 Microbial community differences across groups. A: Relative abundance clustering heatmap. The clustering tree on the left represents species-level clustering. The values in the central heatmap correspond to Z-scores obtained by standardizing the relative abundance of each species. Linear discriminant analysis effect size analysis of species with intergroup differences comprises two parts; B: Linear discriminant analysis score bar chart. The horizontal axis represents group information (diabetes mellitus group, n = 8), diabetes mellitus-colorectal cancer group (n = 11), colorectal cancer group (n = 10), and control group (n = 11). The vertical axis represents species information; C: Evolutionary cladogram. Circles radiating from the inside outward represent taxonomic levels (e.g., genus or species). Each small circle at a given taxonomic level represents a specific taxon, and the diameter of the small circle is positively proportional to the relative abundance. Species with no significant differences are considered as a single group, and biomarkers of different species follow their respective group assignments. Orange nodes indicate microbial groups that play an important role in the orange module, while green nodes represent those important in the green module. The species corresponding to each letter in the cladogram are listed in the legend on the right; D: Krona chart visualizing species annotation results. Circles represent different taxonomic levels (phyla, families, genera, and species) from inside to outside. The size of each fan represents the relative proportion of the corresponding species. DM: Diabetes mellitus; CRC: Colorectal cancer; LDA: Linear discriminant analysis.

Figure 4 Effects of Bolteae supernatant on proliferating cell nuclear antigen expression and proliferation of colorectal cancer cells. A and B: Quantitative polymerase chain reaction analysis of the expression level of proliferating cell nuclear antigen (PCNA) in colorectal cancer cells. There was a significant difference between the intervention group and the non-intervention group, ^aP < 0.05, n = 6. The vertical axis represents the relative abundance of PCNA in RKO (Figure 4A) and LoVo cells (Figure 4B), while the horizontal axis represents different times and intervention concentrations; C and D: Direct cell counting results. RKO colorectal cancer cells (Figure 4C) and LoVo colorectal cancer cells (Figure 4D) were counted at various time points following treatment with different concentrations of Bolteae supernatant; E and F: Cell proliferation measured using the Cell Counting Kit-8 assay. After transfection, absorbance was measured using a fluorescence spectrophotometer. Significant differences (P < 0.05) were observed compared to untreated and other intervention conditions. Spectroscopic measurements of RKO colon cancer cells (Figure 4E) and LoVo colon cancer cells (Figure 4F) at different time points after intervention with various concentrations of Bolteae supernatant are shown. Cells treated with a 1:10 ratio of the supernatant demonstrated the most significant proliferation within 24 hours; G: Representative images taken by an image-based cell counting instrument. Viable cells are distinguished by their larger and more uniform volume, while debris appears smaller and irregularly shaped. Viable cells remain unstained (bright), whereas non-viable cells are stained (dark/blue). The software quantifies the identified viable and non-viable cells and, combined with the known sample volume, automatically calculates the concentration of live cells (Figure 4C and D), accompanied by the display of the captured image. Data were analyzed using analysis of variance.

Figure 5 Functional analysis based on the Kyoto Encyclopedia of Genes and Genomes database. A: Statistical chart of the number of annotated genes in each database. The numbers on the bar chart represent the number of unigenes successfully annotated. The other axis represents the level 1 functional categories within each database; B: Sample clustering analysis based on functional abundance. The left side of the figure displays the clustering tree structure based on Bray-Curtis distance. The right side shows the relative abundance distribution of functions at level 1 for each sample; C: Functional abundance clustering heatmap based on the top 5 most abundant functions per sample, using the functional annotations and abundance data from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The horizontal axis represents sample information, and the vertical axis provides functional annotation information. The clustering tree on the left represents functional clustering. The values in the central heatmap correspond to the Z-scores obtained by normalizing the relative abundance of functions in each row; D: Top 10 KEGG level 1 functional ranked by relative abundance across samples. The Y-axis indicates the proportion of annotations within each functional category, and the X-axis shows the sample names. Relative abundance was calculated at multiple levels across databases. This figure shows the relative abundance distribution at KEGG level 1 as an example; E-G: Dimensionality reduction analysis based on functional abundance in the KEGG database. The more similar the functional composition between samples, the closer their distances in the dimensionality reduction plot. When the stress value of NMDS analysis at level 1 is less than 0.2, the non-metric multidimensional scaling analysis is considered to have a certain degree of reliability (Figure 5E). Principal component analysis at level 1 is shown, where the horizontal axis represents the first principal component, the vertical axis represents the second principal component, and the percentages represent the contribution of each component to sample differences (Figure 5F). Dimensionality reduction analysis based on Bray-Curtis distance derived from species abundance was also performed, as illustrated by principal coordinate analysis at level 1 (Figure 5G). DM: Diabetes mellitus; CRC: Colorectal cancer; PCA: Principal component analysis; NMDS: Non-metric multidimensional scaling; PCoA: Principal coordinate analysis.

Figure 6 Comparison of cancer-related metabolic pathways between the diabetes mellitus and diabetes mellitus-colorectal cancer groups based on Kyoto Encyclopedia of Genes and Genomes database analysis. Differences in metabolic pathway maps between the diabetes mellitus (DM) and DM-colorectal cancer groups were examined using the Kyoto Encyclopedia of Genes and Genomes database, and a metabolic pathway webpage map was generated. The figure highlights cancer-related metabolic pathways within the Kyoto Encyclopedia of Genes and Genomes pathway overview map, showing both shared and unique metabolic pathway information between the two groups. In the diagram, nodes represent chemical compounds, and edges represent enzyme-catalyzed reactions. Red indicates reactions common to both groups, while blue denotes reactions unique to the DM-colorectal cancer group. A: Pathways of colorectal cancer; B: Pathways in cancer.

Figure 7 Effects of Enterocloster bolteae treatment on gene expression in LoVo and RKO colorectal cancer cells. A P value < 0.05 indicates statistically significant differences. All data were normalized to the glyceraldehyde-3-phosphate dehydrogenase endogenous control. Error bars represent standard error of the mean from triplicate experiments. Statistical analysis was performed using one-way analysis of variance with Dunnett’s post-test. Colon cancer cells were treated with Enterocloster bolteae supernatant at a 1:10 ratio (bacterial supernatant to high-glucose Dulbecco’s modified Eagle’s medium) for 24 hours. Compared with controls, relative mRNA expression levels of different targets in LoVo and RKO colorectal carcinoma cell lines were assessed by quantitative real-time polymerase chain reaction. A: 16S rDNA sequence from the Ribosomal Database Project (http://rdp.cme.msu.edu/index.jsp). The comparison results indicate that the strain is Enterocloster bolteae (formerly Clostridium bolteae); B: DNA target band obtained by excising the electrophoresis band of polymerase chain reaction products; C and D: Relative mRNA expression levels of Rho, Rac, hMSH6, Elongin C (ELOC/TCEB1), and TRK in LoVo (Figure 7C) and RKO (Figure 7D) colorectal carcinoma cell lines, as assessed by quantitative real-time polymerase chain reaction.

Figure 8 Silencing of Rac, Rho, hMSH6, and Elongin C via siRNA and assessment of the silencing effect on the proliferation marker proliferating cell nuclear antigen in RKO and LoVo colorectal cancer cells. A: Quantitative real-time polymerase chain reaction quantitative analysis of Rac, Rho, hMSH6, and Elongin C expression levels in RKO cells. The vertical axis represents the relative abundance of these transcripts in colorectal cancer cells, and the horizontal axis represents different intervention conditions (no Bolteae supernatant, or a 1:10 ratio of Bolteae supernatant to culture medium); B: Quantitative real-time polymerase chain reaction quantitative analysis of Rac, Rho, hMSH6, and Elongin C expression levels in LoVo cells; C: Western blot detection of the proliferation marker proliferating cell nuclear antigen (PCNA) in RKO cells transfected with siRNA. The upper panel shows the immunoblot bands, and the lower panel presents the corresponding densitometric quantification. The vertical axis represents the integrated intensity of PCNA, while the horizontal axis indicates different intervention conditions (no Bolteae supernatant, or a 1:10 ratio of Bolteae supernatant to culture medium); D: Western blot detection of the proliferation marker PCNA in LoVo cells transfected with siRNA. PCNA: Proliferating cell nuclear antigen.