De-regulated microRNAs in pediatric cancer stem cells target pathways involved in cell proliferation, cell cycle and development

Abstract

Background: microRNAs (miRNAs) have been implicated in the control of many biological processes and their deregulation has been associated with many cancers. In recent years, the cancer stem cell (CSC) concept has been applied to many cancers including pediatric. We hypothesized that a common signature of deregulated miRNAs in the CSCs fraction may explain the disrupted signaling pathways in CSCs.

Methodology/results: Using a high throughput qPCR approach we identified 26 CSC associated differentially expressed miRNAs (DEmiRs). Using BCmicrO algorithm 865 potential CSC associated DEmiR targets were obtained. These potential targets were subjected to KEGG, Biocarta and Gene Ontology pathway and biological processes analysis. Four annotated pathways were enriched: cell cycle, cell proliferation, p53 and TGF-beta/BMP. Knocking down hsa-miR-21-5p, hsa-miR-181c-5p and hsa-miR-135b-5p using antisense oligonucleotides and small interfering RNA in cell lines led to the depletion of the CSC fraction and impairment of sphere formation (CSC surrogate assays).

Conclusion: Our findings indicated that CSC associated DEmiRs and the putative pathways they regulate may have potential therapeutic applications in pediatric cancers.

Figure 1. Heatmap of miRNA profile in 6 cell lines.

(A). The expression values of 380 DEmiRs were measured and hierarchical clustering was performed. The expression profile of HepG2 and Hep293TT were clustered with Daoy. RD-ES and MG-63 are grouped together with SH-SY5Y. Differentially expressed miRNAs (B). Twenty-six miRNAs with at least 2-fold change in 4 or more cell-lines were selected. Twenty-three miRNAs were up regulated and 3 were down regulated.

Figure 2. p53 signal pathway of KEGG.

(A). The p53 pathway that includes 8 signaling components as defined by KEGG was highly regulated by the CSC associated DEmiRs. Five of the 26 DEmiRs were implicated in this pathway, hsa-miR-21-5p, hsa-miR-135b, hsa-miR-29, hsa-miR-655, hsa-miR-494. TGF-beta pathway of KEGG (B). The TGF-beta pathway was also regulated by the CSC associated DEmiRs. Receptor TGFBR1 was regulated by hsa-miR-101-3p, and receptor TGFBR2 was regulated by hsa-miR-21-5p, and hsa-miR-519a-3p. The receptor BMPR2 was regulated by hsa-miR-101-3p, hsa-miR-21-5p, hsa-miR-181c-5p, and hsa-miR-494. Nodal type II receptor kinase and receptor ACVR2B were also regulated by hsa-miR-101-3p.

Figure 3. hsa-miR-21-5p knockdown on the ALDH^BR fraction in Daoy.

(A). Approximately a two-fold reduction in the fraction enriched for stem-like cells was observed in the hsa-miR-21-5p knockdown. Data shown is representative of three independent experiments. hsa-miR-135b-5p inhibition on the ability to form neurospheres in SK-N-NE(2) (B). An impairment of sphere formation was seen in the SK-N-BE(2) hsa-miR-135b-5p stable knockdown. hsa-miR-135b-5p KD1, KD2 and KD3 were from the same clone. Data represents three independent experiments, presented as the mean ± SEM.