As a marker of organ differentiation and maturation (especially gastric differentiation), PGC has unique physiological and pathological characteristics (10, 11). At present, research on PGC in gastric cancer has mainly focused on its expression level, with its upstream regulatory mechanism yet to be explored.
A ceRNA network mediated by DElncRNA/DEcircRNA plays an important role in the formation and development of tumors and has been used to explore the pathology, diagnosis, and prognosis of different tumors, including gastric cancer (12). In this study, RNA-seq technology was used to detect mRNAs, lncRNAs, and circRNAs based on PGC expression; an enrichment analysis of DEmRNAs was performed and a PPI network was constructed to explore its biological role and potential interacting molecules. A cis lncRNA regulatory network was predicted to analyze its potential role in gastric cancer. The positive correlation between lncRNA–mRNA pairs and circRNA–mRNA pairs was screened and their common target miRNAs were predicted. A ceRNA network was systematically constructed that was co-mediated by PGC expression-related DElncRNAs/DEcircRNAs. The sequencing results were further verified by qRT–PCR using gastric cancer cells and tissues, which formed the basis for further research on the post-transcriptional regulatory mechanisms of PGC in gastric cancer.
RNA sequencing indicated a total of 637 mRNAs, 698 lncRNAs, and 38 circRNAs were differentially expressed, which suggested the association of these RNAs with the expression and function of PGC. Enrichment results showed that DEmRNAs related to PGC expression mainly focused on cell adhesion molecules, and chemokine, oxytocin and cAMP signaling pathways. These pathways were shown to be closely related to the occurrence and development of tumors (13–15). Recently, researchers have claimed that the ceRNA network mediated by non-coding RNA regulated these pathways and thus had an influence on the development of different tumors (16, 17). In other words, PGC regulated and modified the function of these pathways through a ceRNA network mediated by PGC expression–related DElncRNAs and DEcircRNAs, thus leading to a regulatory role in gastric cancer cells at the post-transcriptional level. The PPI network of DEmRNAs was made up of 503 nodes and 1179 edges. A direct interaction relationship exists between PGC–CFH (interaction score = 0.458), PGC–PPARG (interaction score = 0.452), and PGC–MUC6 (interaction score = 0.429). The qRT–PCR validation in vivo and in vitro indicated that only the differential expression of PPARG was statistically significant, which is in the same direction as PGC. Spearman correlation analysis also indicated that PGC was positive with PPARG (r = 0.276, P = 0.009). PPARG is a type of nuclear hormone receptor that can regulate various cell functions, including lipogenesis, lipid biosynthesis, energy consumption and storage, and inflammation (18). The study found that PPARG also promoted epithelial cell differentiation and inhibited tumor cell proliferation (19, 20), which is consistent with the conclusion that PGC is a marker of organ differential maturation. Such conclusions suggested that PGC may interact with PPARG to co-regulate the differential maturation of gastric cancer cells, thus affecting tumor progression. But the specific mechanism still needs to be further explored.
To understand the potential roles of PGC expression–related lncRNAs, adjacent target genes were predicted to construct a cis-regulatory network. A total of 105 differentially expressed cis-regulatory genes were found within 10 kbp upstream and downstream of 94 DElncRNAs, which constituted 181 cis lncRNA–gene pairs. The cis-regulation function of non-coding RNA might participate in various biological processes (21) while the cis-regulation function of lncRNA might affect tumor progression (22, 23). Our analysis indicated that DElncRNAs were mainly positive with adjacent protein-coding genes, with a negative correlation being relatively few in number. Therefore, we speculated that PGC expression–related DElncRNAs mainly positively regulated the expression of adjacent protein-coding genes. In addition, a ceRNA network mediated by DElncRNAs was constructed by 19 DElncRNAs, 85 DEmRNAs, and 76 miRNAs, of which HCG18, SNHG16, and SNHG1 showed the highest degree scores. Most research indicated that ceRNA mediated by DElncRNA regulated the progression of gastric cancer, colorectal cancer, and osteosarcoma (24–26). HCG18 was shown to promote gastric cancer progression by up-regulating DNAJB12 via miR-152-3p (27). SNHG1 facilitated the growth and migration of gastric cancer cells via the miR-140/ADAM10 axis (28). This study initially found that these lncRNAs were associated with PGC expression and its mediated ceRNA network might have affected gastric cancer progression.
A ceRNA network mediated via PGC expression–related DEcircRNAs was constituted to explore a potential role. It was found that 21 DEcircRNAs, 32 DEmRNAs, and 43 miRNAs constituted a ceRNA network mediated via DEcircRNAs. Of these, the top three with the highest degree score of DEcircRNAs included hsa_circ_0031583, hsa_circ_0008197, and hsa_circ_0036627. A ceRNA network mediated via DEcircRNAs modulated histological classifications and gastric cancer progression (29) and the stemness properties of colorectal cancer stem cells (30). No report has shown that hsa_circ_0031583, hsa_circ_0008197, and hsa_circ_0036627 affected tumor progression. Using bioinformatics, this study initially revealed that the ceRNA networks mediated by PGC expression–related hsa_circ_0031583, hsa_circ_0036627, and hsa_circ_0036627 may play a major role in the progression of gastric cancer. This provides important clues for subsequent research of mechanisms in gastric cancer.
Based on a ceRNA network mediated by DElncRNAs and DEcircRNAs, a total of 11 DElncRNAs, 13 circRNAs, and 35 miRNA–mRNA pairs were used to construct their co-mediated ceRNA network. As is well known, ceRNA networks mediated via non-coding RNAs play an important role in gastric cancer (31–33). In this study, the co-regulated network included the SNHG16/hsa_circ_0008197–hsa-mir-98-5p/hsa-let-7f-5p/hsa-let-7c-5p–PGC axis. This was directly related to PGC and was of great significance in exploring its post-transcriptional regulatory mechanisms in gastric cancer. Therefore, qRT–PCR validation for SNHG16 and hsa_circ_0008197 was performed in vivo and in vitro. It was found that SNHG16 and hsa_circ_0008197 were differentially expressed at the gastric cell and tissue levels, and were positive for PGC, respectively (SNHG16: r = 0.35, P = 0.002; hsa_circ_0008197: r = 0.346, P = 0.005). This suggested the presence of a ceRNA network directly related to PGC. Small nucleolar RNA host gene 16 (SNHG16) was encoded by a 7571-bp region on chromosome 17q25.1 and was considered as a cancer-related lncRNA (34). A previous study showed that SNHG16 sponged miRNA by binding to MREs to modulate the expression of tumor-related target genes, thus regulating the proliferation, apoptosis, migration and invasion of tumor cells (35). A dual luciferase reporter experiment and RNA binding protein immunoprecipitation were used to show that SNHG16 sponged hsa-mir-98-5p to regulate the progression of osteosarcoma (36). SNHG16 promoted the development of bladder cancer via a miR-98/STAT3/Wnt/β-catenin pathway (37). No report exists of SNHG16 sponging hsa-let-7c-5p/hsa-let-7f-5p to modulate tumor progression, but several investigations showed that hsa-let-7c-5p and hsa-let-7f-5p also regulated proliferation, migration, and invasion of tumor cells (38–40). This study initially found and validated the SNHG16–mir-98-5p/hsa-let-7f-5p/hsa-let-7c-5p–PGC axis as having an influence on gastric cancer progression. hsa_circ_0008197 is located on chr1:51032749–51061888. Although research on the association of hsa_circ_0008197 with human diseases is lacking, our study initially found and verified that hsa_circ_0008197 and SNHG16 co-mediated a PGC-related ceRNA network to transform the SNHG16/hsa_circ_0008197–mir-98-5p/hsa-let-7f-5p/hsa-let-7c-5p–PGC axis. We speculated that this network might cause a change in PGC expression to generate a biological effect via a systemic post-transcriptional regulatory mechanism, thus influencing tumor progression. This yields a novel notion related to PGC pathogenetic mechanisms in gastric cancer.
In conclusion, this study found that the expression of many types of mRNAs, lncRNAs, and circRNAs are related to PGC expression, which, in turn, is involved in cancer-related pathways. DElncRNA related to PGC expression might influence tumor progression by positively regulating the expression of adjacent protein-coding genes. DElncRNAs and DEcircRNAs related to PGC expression co-mediated a complicated ceRNA network to modulate its expression, of which the closest network is SNHG16/hsa_circ_0008197-mir-98-5p/hsa-let-7f-5p/hsa-let-7c-5p-PGC. This study contributes to a foundation for the subsequent exploration of possible regulatory mechanisms related to PGC in gastric cancer.