Time-dependent gene expression alterations in the MKN74 gastric cancer cell line treated with E. faecalis
To determine the gene expression alterations between control day 1, infected day 1and infected day 5 groups of human gastric carcinoma MKN74 cells all genome expression data analyzed by lineer regression. Based on the results, 138 genes corresponding to 165 probesets with a standard deviation value above 1.0, P-value below 0.05, and Pearson R value above 0.99 showed statistically significant expression alteration. We focused on these genes that altered variation in expression between groups for further analyzes. 10 probesets were positively correlated and upregulated and 155probesets were negatively correlated and downregulated in the time depended E. faecalis treated groups (Additional File 1).
Our hierarchical cluster analysis demonstrated gene alterations between 3 certain groups; control day 1, infected day 1 and infected day 5. Results defining as 155 of the probesets were negatively correlated, high expressed in the control day 1 group and expressions are decreases at infected day 1 group and so on at infected day 5 group. And conversely 10 of the probesets were positively correlated, low expressed in the control day 1 group and expressions are increases at infected day 1 and so on at infected day 5 group (Fig. 1). So, there were significantly altered genes between control and treated groups.
Gene alterations due to treatment with E. faecalis
In order to determine whether this expression change was caused by E. faecalis or cancer itself, varience analyzes and t-test were performed between control day 5 group and infected day 5 group 138 genes/165 probesets expression data. To better analyze gene expression alteration, long range group “infected day 5” group was selected for variance analysis and t-test. Thus, 12 statistically significant genes (NDC80, NCAPG, CENPA, KIF23, BUB1B, BUB1, CASC5, KIF2C, CENPF, SPC25, SMC4, KIF20A) corresponding 15 probesets with a standard deviation above 1.0 and a T-test p-value less than 0.01 were detected and selected (Table 1) (Additional File 2).
Table 1
The list of 12 genes (15 Probe Sets) which have the most alterations in expression. These genes have standard deviation above 1.0 and T-test value less than 0.01 between 5 days control group and 5 days treated group. These significant values indicate that the change occurred due to infection.
Probe Set | Gene Symbol | St Dev | T-Test |
204162_at | NDC80 | 1.76016363 | 0.00153695 |
218663_at | NCAPG | 1.74829872 | 0.0002009 |
204962_s_at | CENPA | 1.5109911 | 0.00039232 |
218662_s_at | NCAPG | 1.46733952 | 0.00062378 |
204709_s_at | KIF23 | 1.42573476 | 0.00046214 |
203755_at | BUB1B | 1.36025723 | 1.7849E-05 |
215509_s_at | BUB1 | 1.30655845 | 0.00019534 |
209642_at | BUB1 | 1.26836983 | 0.0001898 |
228323_at | CASC5 | 1.19910534 | 4.6314E-05 |
211519_s_at | KIF2C | 1.17437172 | 0.00024729 |
207828_s_at | CENPF | 1.13683208 | 8.8436E-05 |
209891_at | SPC25 | 1.13377039 | 0.00260856 |
201664_at | SMC4 | 1.05623047 | 0.00770427 |
218755_at | KIF20A | 1.02361512 | 0.0033544 |
209408_at | KIF2C | 1.02269745 | 0.00046939 |
All of these 12 genes were found negatively correlated and downregulated in the linear regression analysis.
Figure 2 shows the expression alterations of these 12 genes time-based between control day 1, infected day 1 and infected day 5 (Fig. 2). Gene expressions have been shown to decrease as the infection process progresses in all these 12 genes.
Network construction and identification of key candidate genes using gene-gene interaction
Network analysis was carried out with Cytoscape to better show the biological connection of these 12 genes, which decreased in expression from day 1 to day 5 during infection. As understood from the figure, our determined 12 genes were shown to have a strong network relationship between each other and also with other candidate genes (Fig. 3). The linking line between genes illuminate the network of these genes. The thickness of the linking line determines the power of connection of the related genes. For instance, the linking line between NCAPG and SMC4 represent one of the thickest lines. This indicates the link formed between these genes has been determined to be stronger by studying more clearly. Additionally, the black nodes indicate the target genes giving by authors. On the other hand, the gray nodes demonstrate the genes which associated genes determined by GeneMANIA application. The size of nodes reversely correlated with score. For example, NDC80 gene has virtually 0,56 score and the other gene, CENPT’s score is 0,05. This directly correlated with the size of the nodes. NDC80 has higher node size than CENPT.
Functional Enrichment Of Genes And Correlations With Pathways
To reveal the relationship of these 12 genes with cellular functions, pathways and to understand the new significance biological processes pathway analysis was done by DAVID software. 4 important pathways cell cycle, pyrimidine metabolism, mismatch repair, P53 signaling pathway have been identified which associated with our 12 genes that were significant during the exposure to E. faecalis. The relationships of these pathways with cancer development is very clear such as importance of cell cycle in the gastric cancer cells (Table 2).
Table 2
Pathways related to the genes are linked. It is seen that important pathways in cancer progression are related to our genes. Most of these genes are linked to the cell cycle pathway from the database for annotation, visualization and integrated discovery (DAVID).
Category | Pathways | Count | P-Value | Benjamini |
KEGG_PATHWAY | Cell Cycle | 6*1 | 1,3E-3 | 9,0E-2 |
KEGG_PATHWAY | Pyrimidine metabolism | 2*2 | 3,1E-3 | 1,1E-1 |
KEGG_PATHWAY | p53 signaling pathway | 4*3 | 1,0E-2 | 1,7E-1 |
KEGG_PATHWAY | Mismatch repair | 3*4 | 9,1E-3 | 2,0E-1 |
*1NDC80, NCAPG, BUB1B, BUB1, CENPA, KIF23. *2BUB1B, BUB1. *3KIF2C, SPC25, KIF20A, CENPF. *4SMC4, CENPA, NCAPG.
The significantly enriched gene sets and their ES, NES, NOM p-value, FWER p-value, FDR q-value represented in Additional File 3. According to GSEA, the cell cycle checkpoint as well as mitotic cell cycle checkpoint gene set were found as significantly correlated with the genes that present in the data. These gene sets even correlated with our pathway analysis in Table 2 and other emphasized studies. The enriched genes in these two gene sets were NCD80, BUB1, BUB1B, CENPF. Furthermore, Fig. 4 was demonstrating the cell cycle checkpoint gene set graph. This graph showed the genes which were NCD80, BUB1, BUB1B, CENPF enriched in control day 5 group that also referred as positively correlated. On the other hand, the same genes negatively correlated in infected day 5 group by down regulating. The same outcomes occurred for mitotic cell cycle checkpoint gene set (Fig. 5).