Epigenetic Field Cancerization in Gastric Cancer: microRNAs as Promising Biomarkers

Abstract

Background: The biological role of microRNAs (miRNAs) in field cancerization is unknown. To investigate the involvement of miRNAs in gastric field cancerization, we evaluated the expression profile of ten miRNAs and their diagnostic value. Methods: We used three groups of FFPE gastric samples: non-cancer (NC), cancer adjacent (ADJ) and gastric cancer (GC). The expression profiles of hsa-miR-10a, -miR-21, -miR-29c, -miR-135b, -miR-148a, -miR-150, -miR-204, -miR-215, -miR-483 and -miR-664a were investigated using qRT-PCR. The results obtained by qRT-PCR were validated in Small RNA-Seq data from the TCGA database. The search for target genes of the studied miRNAs was performed in the miRTarBase public database and miRTargetLink tool, using experimentally validated interactions. In addition, we also performed the functional analysis of these genes using enrichment in KEGG pathways. The potential as biomarker was evaluated using a receiver operating characteristic (ROC) curve and the derived area under the curve (AUC>0.85) analysis. Results: The miRNAs hsa-miR-10a, -miR-21, -miR-135b, hsa-miR-148a, -miR-150, -miR-215, -miR-204, -miR-483 and -miR-664a were up-regulated in ADJ and GC compared to NC (P<0.03); and hsa-miR-21 and -miR-135b were up-regulated in GC compared to ADJ (P<0.01). Hsa-miR-148a, -miR-150, -miR-215, -miR-483 and -miR-664a were not differentially expressed between GC and ADJ, suggesting that both share similar changes (P>0.1). The TS-miR hsa-miR-29c was up-regulated in ADJ compared to NC and GC (P<0.01); we did not observe a significant difference in the expression of this miRNA between NC and GC. This feature may be an antitumor mechanism used by cancer-adjacent tissue because this miRNA regulates the BCL-2, CDC42 and DMNT3A oncogenes. The expression level of hsa-miR-204 was associated with Helicobacter pylori infection status (P<0.05). Functional analysis using the genes regulated by the studied miRNAs showed that they are involved in biological pathways and cellular processes that are critical for the establishment of H. pylori infection and for the onset, development and progression of GC. hsa-miR-10a, -miR-21, -miR-135b, -miR-148a, -miR-150, -miR-215, -miR-483 and -miR-664a were able to discriminate NC from other tissues with great accuracy (AUC>0.85). Conclusion: The studied miRNAs are closely related to field cancerization, regulate genes important for gastric carcinogenesis and can be potentially useful as biomarkers in GC.

Keywords: biomarker; epigenetic; field cancerization; gastric cancer; miRNA.

Figure 1

Expression levels of the studied miRNAs among the non-cancer, cancer-adjacent and gastric cancer groups. A) hsa-miR-10a; B) hsa-miR-21; C) hsa-miR-29c; D) hsa-miR-135b; E) hsa-miR-148a; F) hsa-miR-150; G) hsa-miR-204; H) hsa-miR-215; I) hsa-miR-483; J) hsa-miR-664a. NC = Non cancer; ADJ = Adjacent to gastric cancer; GC = Gastric cancer. *P value < 0.05; **P value < 0.001; ***P value < 0.0001. P value adjusted by Bonferroni's correction.

Figure 2

Heatmap of normalized expression of the ten studied miRNAs. The unsupervised hierarchical clustering showed that the miRNAs studied were able to separate non-cancer from cancer-adjacent and gastric cancer tissues.

Figure 3

Evaluation of the discriminatory performance of eight miRNAs. These eight miRNAs were able to discriminate the non-cancer from the other tissues with great accuracy. The area under the curve (AUC>0.85).

Figure 4

Influence of H. pylori infection on hsa-miR-204 expression. *P value<0.05. P value adjusted by Bonferroni's correction.

Figure 5

Functional enrichment in KEGG pathways using the target genes of the studied miRNAs. In highlight pathways relevant for establishment and progression of GC. A) Enrichment using driver genes. Information based on table SA2 in Vogelstein et al. . B) Enrichment using genes provided by the miRTargetLink tool.

Figure 6

Functional enrichment in KEGG pathways using the target genes of the five miRNAs associated to H. pylori infection. In highlight some KEGG pathways relevant for establishment and progression of infection.