ABSTRACT: Osteosarcoma (OS) is a primary bone tumor with a high incidence and mortality in children and adolescents. Emerging evidence shows that microRNAs (miRNAs) participate in biological tumor mechanisms by targeting downstream messenger RNAs (mRNAs). This article aimed to investigate the potential regulatory targets of microRNA-199a-3p (miR-199a-3p) in OS and to contribute to the understanding of miR-199a-3p-related OS regulatory mechanisms. MicroRNA-related Gene Expression Omnibus (GEO) chips, ArrayExpress chips and literature data were used to determine the expression of miR-199a-3p in OS and pooled to explore its potential clinical value. To investigate the target genes of miR-199a-3p further, we integrated the results from the following three-part gene study: Twelve online prediction tools were used to predict the target genes of miR-199a-3p; the GEO GSE89370 chip transfected with miRSelect pEP-miR-199a-3p was used to analyze the downregulated differentially expressed genes (DEGs) in OS cells; and highly expressed DEGs were derived from an in-house microarray generated from three pairs of clinical OS and normal tissue samples acquired through our department. Then, we analyzed the target genes using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases and the protein-protein interaction (PPI) network to further identify the primary target genes. In addition, we constructed transcription factor (TF)-miRNA-joint gene feed-forward regulatory loops (FFLs) with Circuits DB using miR-199a-3p as the core. A comprehensive meta-analysis of a hub of miR-199a-3p targeted genes was performed to integrate expression level, summary ROC (sROC) curves and survival analysis results from the GEO data for verification and exploration. Finally, the expression levels of the hub genes were verified in OS tissues and U2OS cells by immunohistochemistry (IHC) and immunocytochemistry (ICC). Data on miR-199a-3p expression were obtained from three data sets (GSE65071, GSE69524, and PMID 21666078), which showed low miR-199a-3p expression levels in OS tissues. The combined data indicated the same tendency, with the SMD of the random effect model, as shown in forest plots, being -2.8 (95% CI: -4.49, -1.11). In addition, we determined that miR-199a-3p may serve as a molecular marker useful for distinguishing OS tissues from normal tissues with high sensitivity and specificity, with the measured outcomes being 0.94 (95% CI: 0.80, 0.99) and 0.96 (95% CI: 0.78, 1.00), respectively. In addition, 391 genes were considered targets of miR-199a-3p in OS, and the enrichment analysis indicated that these targets were mainly enriched in proteoglycans in cancer and in spliceosomes. Four genes, CDKI, CCNB1, AURKA and NEK2, were regarded as hub targets based on the PPI data. Subsequently, TF-miRNA-joint genes FFLs were constructed in Circuits DB and included 17 TFs and 82 joint targets. These joint targets were mainly enriched in spliceosomes. UBE2D1 and RBM25 were regarded as hub joint targets based on the enrichment analysis. All selected target genes were further verified to ensure that they were upregulated in OS and to determine their prognostic significance. At the experimental verification level, the CDK1 protein was confirmed to be positively expressed in the cytoplasm of OS tissues and the U2OS cell line. Our study verified that miR-199a-3p was obviously downregulated in OS. CDK1, CCNB1, NEK2, AURKA, UBE2D1 and RBM25 were identified as potential target genes of miR-199a-3p in OS.
