ACC is a rare aggressive type of endocrine cancer that originates in adrenal cortical cells, whose prognosis is poor(3). At present, the molecular mechanism of ACC development has not yet been elucidated, which brings great challenges to the clinical diagnosis and treatment of ACC. Recent studies have reported that ncRNAs, including miRNAs and lncRNAs, play important roles in cancer initiation and progression(31–35). After the first proposal of ceRNA hypothesis by Salmena et al(22), in which lncRNA competes with mRNA to bind miRNA to form a sponge-like structure, buffering and reducing the ability of miRNA to interfere with the protein encoded by the target gene. Increasing studies about ceRNAs in human cancers have been reported. For example, Long Noncoding RNA (lncRNA)-Mediated Competing Endogenous RNA Networks Provide Novel Potential Biomarkers and Therapeutic Targets for Colorectal Cancer(36). LncRNA-CDC6 promotes breast cancer progression and function as ceRNA to target CDC6 by sponging microRNA-215(37). LncRNA XLOC_006390 facilitates cervical cancer tumorigenesis and metastasis as a ceRNA against miR-331-3p and miR-338-3p(38). LncRNA HOTAIR participates in the development and progression of adrenocortical carcinoma via regulating cell cycle(39). However, some analysis for ceRNAs in adrenocortical carcinoma is still not enough. In this study, a novel mRNA-miRNA-lncRNA triple regulatory network was constructed and each RNA in this network significantly associated with prognosis in adrenocortical carcinoma.
In this study, we identified a total of 325 significant DEGs, consisting of 49 upregulated and 276 downregulated DEGs by searching GEO database, and analyzing two datasets, GSE12368 and GSE19750. GO is widely used as functional enrichment analysis for a large number of genes(40). The results of these significant DEGs related GO analysis showed that they were significantly enriched in some GO terms that were associated with cancer biological behaviors, including cell division, cell adhesion, regulation of signal transduction by p53 class mediator. KEGG pathway enrichment analysis revealed that the significant DEGs were significantly enriched in some cancer-associated pathways, such as pathways in Cell cycle, p53 signaling pathway and Progesterone-mediated oocyte maturation, Proteoglycans in cancer.
To further analyze the relationships and functions of significant DEGs in adrenocortical carcinoma, we used STRING database and obtained PPI networks. It has been widely acknowledged that genes with more node degree in the PPI network usually play more roles. Therefore, we screened the hub genes in the two PPI networks according to node degree. The top ten upregulated and downregulated hub genes were selected for further expression and survival analyses to identify key genes in adrenocortical carcinoma. The analytic results showed that 9 upregulated (CDK1, CCNB1, MCM4, AURKA, NCAPG, RRM2, TYMS and TPX2) hub genes may act as the key genes in adrenocortical carcinoma because alteration was significantly associated with worse prognosis.
MiRNAs and lncRNAs, are involved in regulation of gene expression and function by ceRNA mechanism as previously described. So the upstream miRNAs of the key genes were first predicted. Survival analysis revealed that 4 miRNAs (miR-212-3p, miR-24-3p, let-7a-5p and miR-196a-5p) significantly influenced prognosis in cancer. The tumor suppressive roles of the 4 miRNAs have been reported. For example, Long Noncoding RNA KCNQ1OT1 Accelerates the Progression of Ovarian Cancer via MicroRNA-212-3/LCN2 Axis(41). Consistently, ectopic expression of miR-24-3p suppressed the cell migration, invasion, and proliferation of MCF7, Hep3B, B16F10, SK-Hep1, and PC-3 cells by directly targeting p130Cas(42). Moreover, let-7a-5p inhibited BCL2L1 expression and suppressed lung cancer cell proliferation, migration, and invasion(43). LncRNA NEAT1 promotes colorectal cancer cell proliferation and migration via regulating glial cell-derived neurotrophic factor by sponging miR-196a-5p(44). Then, we further predicted 185 upstream lncRNAs of these key miRNAs. By combining expression analysis and survival analysis for these lncRNAs in adrenocortical carcinoma using GEPIA, only 1 lncRNAs (HOXA11-AS) were defined as the key lncRNAs. The tumor promoted roles of the HOXA11-AS have been reported. For example, LncRNA HOXA11-AS Promotes Proliferation and Invasion of Gastric Cancer by Scaffolding the Chromatin Modification Factors PRC2, LSD1, and DNMT1(45). LncRNA HOXA11-AS promotes proliferation and invasion by targeting miR-124 in human non-small cell lung cancer cells(46). HOXA11-AS promotes the growth and invasion of renal cancer by sponging miR-146b-5p to upregulate MMP16 expression(47). Thus, a mRNA-miRNA-lncRNA network in adrenocortical cancer was successfully established. In this network, some pairs have been identified in some cancers. For example, RRM2-let-7a-5p-SNHG16/MAL2 as key ceRNA subnetwork associated with prognosis of breast cancer(48). MiR-31-5p acts as a tumor suppressor in renal cell carcinoma by targeting cyclin-dependent kinase 1 (CDK1)(49). These reports further indicate the accuracy of our current analytic results. Of course, although attractive findings have been obtained by a series of bioinformatic analyses in our current study, more laboratory experiments need to be performed in the future.