Identification of PGC-related ncRNAs and their relationship with the clinicopathological features of Gastric Cancer

Abstract

Pepsinogen C (PGC) is considered to be the final product of mature differentiated gastric mucosa. The expression level of PGC in gastric mucosa is clearly decreased upon the development of gastric cancer (GC). However, the mechanism behind PGC's down-regulation remains unclear and needs to be clarified. This study aimed to identify PGC-related ncRNAs with the potential to be PGC post-transcriptional regulators and to further explore the association between these ncRNAs and the clinicopathological parameters of GC. Bioinformatic software was used to predict miRNAs binding specifically to PGC and circRNAs binding specifically to these candidate miRNAs. Dual-luciferase reporter assay was performed to validate the completely complementary pairing of PGC and PGC-related ncRNAs. qRT-PCR was applied to determine the expression levels of PGC and PGC-related ncRNAs in GC tissue. hsa-let-7c was predicted to bind to the PGC gene, which was confirmed by dual-luciferase reporter assay. hsa_circ_0001483 and hsa_circ_0001324 were identified to bind to hsa-let-7c by bioinformatic analysis and dual-luciferase reporter assay. In addition, the hsa_circ_0001483/hsa_circ_0001324 -hsa-let-7c-PGC axis was confirmed in tissue by qRT-PCR. The expression level of hsa_circ_0001483 was correlated with peritumoral inflammatory cell infiltration and lymphatic metastasis. hsa_circ_0001483, hsa_circ_0001324, and let-7c were newly identified and validated as PGC-related ncRNAs and showed associations with the clinicopathological features of GC. The hsa_circ_0001483/hsa_circ_0001324-hsa-let-7c-PGC axis in GC may account for the down-regulation of PGC in GC tissue.

Keywords: CircRNA; Gastric cancer; MiRNA; Pepsinogen C; ceRNA network.

Figure 1

MiRNAs targeted to PGC in dual-luciferase reporter assay. Hsa-miR-520a, hsa-let-7c and hsa-miR-98 was significantly down-regulated in PGC wild-type group when compared with the PGC mutant-type (“**” means p < 0.010, “*” means p < 0.050).

Figure 2

The expression level of hsa-let-7c when confected with PGC over-expression plasmid in AGS, HGC-27, and MKN-45 cells (“*” means p < 0.050; “ns” means no difference).

Figure 3

CircRNAs targeted to hsa-let-7c in dual-luciferase reporter assay. A, D. There was no significant reduction in luciferase reporter activity when compared hsa_circ_0001614-WT type and hsa_circ_0001051-WT type with control group. B, C. A significant reduction of luciferase reporter activity was observed in hsa_circ_0001483 and hsa_circ_0001324 wild-type reporter gene group than control group (“***” means p < 0.001, “**” means p <0.010).

Figure 4

Hsa-let-7c was up-regulated in 30 pairs GC tissues (“**” means p < 0.010).

Figure 5

Hsa_circ_0001483, hsa_circ_0001324 and PGC was down-regulated in 66 pairs GC tissues (“***” means p < 0.001). The higher ΔCt value indicates a lower expression.