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2019 ; 11
(12
): 7538-7554
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Exploration and validation of downregulated microRNA-199a-3p, downstream
messenger RNA targets and transcriptional regulation in osteosarcoma
#MMPMID31934299
Huang WT
; Liu AG
; Cai KT
; He RQ
; Li Z
; Wei QJ
; Chen MY
; Huang JY
; Yan WY
; Zhou H
; Chen G
; Ma J
Am J Transl Res
2019[]; 11
(12
): 7538-7554
PMID31934299
show ga
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.
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