As we know, PGC is a key gene in the epithelial differentiation process of stomach, and the regulatory mechanism involved in the significant downregulation of PGC expression in GC still needs in-depth investigation. In the present study, we predicted miRNAs with target binding sites to PGC gene as well as the target circRNAs for the miRNA. Then the dual-luciferase reporter experiments were performed to confirm the targeted binding relationships between PGC and miRNAs, miRNAs and circRNAs. And the expression patterns of PGC and PGC-related ncRNAs in GC tissues have also been verified by qRT-PCR. Finally, let-7c, hsa_circ_0001483 and hsa_circ_0001324 were identified as PGC-related ncRNAs in GC and the relationship between these ncRNAs and clinicopathological characteristics of GC was also revealed from the study.
Dysregulation of ncRNAs have been recognized as the novel molecular signatures that indicated the occurrence and development of cancer. As a kind of ncRNAs, miRNAs play an important role in down-regulating the transcription of target mRNAs. Relationship between miRNAs and GC has been reported in the previous studies [25]. For example, miR-92a-1-5p could increase CDX2 by targeting FOXD1 and mediate gastric intestinal metaplasia [26]. MiR-143-3p may inhibit GC growth and be more sensitive to cisplatin by targeting BRD2 [27]. Our present study focused on finding the ncRNAs that regulated the low expression of PGC in GC tissues. The results of dual-luciferase reporter assay showed that 3 of the miRNAs predicted by the software were verified to bind to PGC (hsa-miR-520a, hsa-let-7c and hsa-miR-98). According to previous studies, hsa-miR-520 was demonstrated to play role in regulating proliferation and migration of lung cancer and modulating progression of renal carcinoma while there was no evidence about this miRNA and GC [28, 29]]. And miR-98 could be served as biomarker for prostate cancer diagnosis and it may up-regulated in GC, which was involved in the development and progression of GC [30, 31]. Let-7 miRNA family played a central role in the regulatory relationships between miRNAs and mRNAs in GC, and hsa-let-7c was associated with the clinicopathological parameters of GC [32]. The above three miRNAs were first reported to be related to PGC tissue expression in the present study. And our preliminary research results have reported that the serum hsa-let-7c expression level was inversely related to PGC [33]. These PGC-related miRNAs may be involved in the development of GC by regulating the PGC expression while the specific mechanisms still need to be illuminated. In addition, these miRNAs may act as potential biomarkers for GC.
CircRNA was another type of ncRNAs, whose roles in cancers remained to be further investigated. CircRNAs have been demonstrated to act as miRNAs sponges in a variety of cancer types and may potentially become new biomarkers for cancers [34–36]. Many researches have reported that circRNAs played regulatory roles in GC. Rong et al have found that circHECTD1 activated β-catenin/c-Myc signaling by adsorption of miR-1256, and facilitated glutaminolysis, and thus promoting GC progression [37]. CircAKT3 was found to enhance cisplatin resistance in GC by suppressing hsa-miR-198 to up-regulate PIK3R1 [38]. In this study, we predicted the target circRNAs for hsa-let-7c by bioinformatics methods and these 12 candidates were also validated by qRT-PCR in 30 pairs of GC tissues. We selected four down-regulated circRNAs (hsa_circ_0001483, hsa_circ_0001324, hsa_circ-0001614 and hsa_circ_0001051) (p༜0.001) for the dual-luciferase report assay with hsa-let-7c. The results suggested that hsa_circ_0001483 and hsa_circ_0001324 could combine with hsa-let-7c. Considering the results of dual-luciferase reporter assay, we constructed PGC-specific ceRNA network including hsa_circ_0001483/ hsa_circ_0001324, hsa-let-7c and PGC in GC. According to the results, we supposed that hsa_circ_0001483 and hsa_circ_0001324 could down-regulate PGC expression by competitively binding with hsa-let-7c and these two unreported circRNAs may also serve as biomarkers for diagnosis of GC.
In this study, the expression level of hsa-let-7c, hsa_circ_0001483, hsa_circ_0001324 and PGC in GC tissues and distant normal tissues were further investigated by qRT-PCR. We found that hsa_circ_0001483 and hsa_circ_0001324 was significantly down-regulated in GC tissues and have good collinear relationship with PGC expression which indicated that the low expression of the two circRNAs coincides with PGC low expression in GC. In addition, hsa-let-7c was significantly overexpressed in GC tissues. The above results suggested the possible endogenous interaction between these ncRNAs and gene PGC, that is, the abnormally low expression of hsa_circ_0001483 and hsa_circ_0001324 as well as high expression of hsa-let-7c in GC tissues causes more hsa-let-7c bind to PGC-UTR region, thus down-regulating the expression of PGC. Beyond that, hsa_circ_0001483 expression level was associated with peritumoral inflammatory cell infiltration level and lymphatic metastasis. The immune cells infiltration and metastasis are key events that should be considered in evaluating GC prognosis and therapeutic strategy [39–41]. All these results suggested that the dysregulation of hsa_circ_0001483 may influence the immune response, chemosensitivity-related features and metastasis situation of GC patients, which may serve as potential biomarker for GC prognosis. In addition, we found that the higher expression of hsa_circ_0001324 and hsa_circ_0001483 suggested the trend of better OS (p = 0.070 and p = 0.081, respectively). Combined with the results of previous study, the low PGC expression level was correlated with shorter OS in GC patients [42], we hypothesized that low expression of hsa_circ_0001483 and hsa_circ_0001324 attenuated their ability to absorb hsa-let-7c, thereby down-regulating the expression of PGC, which may affect the prognosis of GC patients.