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Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis
Published on: March 30, 2019
Integrated approaches to miRNAs target definition: time-series analysis in an osteosarcoma differentiative model
A Grilli1, M Sciandra2,3, M Terracciano4
1Laboratory of Experimental Oncology, CRS Development of Biomolecular Therapies, Rizzoli Orthopedic Institute, Via di Barbiano 1/10, 40136, Bologna, Italy. andrea.grilli@ior.it.
Background:
microRNAs (miRs) are small non-coding RNAs involved in the fine regulation of several cellular processes by inhibiting their target genes at post-transcriptional level. Osteosarcoma (OS) is a tumor thought to be related to a molecular blockade of the normal process of osteoblast differentiation. The current paper explores temporal transcriptional modifications comparing an osteosarcoma cell line, Saos-2, and clones stably transfected with CD99, a molecule which was found to drive OS cells to terminally differentiate.
Methods:
Parental cell line and CD99 transfectants were cultured up to 14 days in differentiating medium. In this setting, OS cells were profiled by gene and miRNA expression arrays. Integration of gene and miRNA profiling was performed by both sequence complementarity and expression correlation. Further enrichment and network analyses were carried out to focus on the modulated pathways and on the interactions between transcriptome and miRNome. To track the temporal transcriptional modification, a PCA analysis with differentiated human MSC was performed.
Results:
We identified a strong (about 80 %) gene down-modulation where reversion towards the osteoblast-like phenotype matches significant enrichment in TGFbeta signaling players like AKT1 and SMADs. In parallel, we observed the modulation of several cancer-related microRNAs like miR-34a, miR-26b or miR-378. To decipher their impact on the modified transcriptional program in CD99 cells, we correlated gene and microRNA time-series data miR-34a, in particular, was found to regulate a distinct subnetwork of genes with respect to the rest of the other differentially expressed miRs and it appeared to be the main mediator of several TGFbeta signaling genes at initial and middle phases of differentiation. Integration studies further highlighted the involvement of TGFbeta pathway in the differentiation of OS cells towards osteoblasts and its regulation by microRNAs.
Conclusions:
These data underline that the expression of miR-34a and down-modulation of TGFbeta signaling emerge as pivotal events to drive CD99-mediated reversal of malignancy and activation of differentiation in OS cells. Our results describe crucial and specific interacting actors providing and supporting their relevance as potential targets for therapeutic differentiative strategies.
Insights
MicroRNAs (miRs) regulate gene expression. In osteosarcoma cells, miR-34a and TGFbeta signaling modulation drive cancer cell differentiation, offering potential therapeutic targets.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- MicroRNAs (miRs) are small non-coding RNAs regulating gene expression post-transcriptionally.
- Osteosarcoma (OS) is linked to blocked osteoblast differentiation.
- CD99 expression induces terminal differentiation in OS cells.
Purpose of the Study:
- To explore temporal transcriptional modifications in osteosarcoma cells.
- To compare gene and microRNA expression in Saos-2 cells and CD99-transfected clones.
- To investigate the role of CD99 in osteosarcoma differentiation.
Main Methods:
- Culturing parental and CD99-transfected OS cells in differentiating medium.
- Profiling gene and microRNA expression using arrays.
- Integrating gene and microRNA data through sequence complementarity and expression correlation.
- Performing enrichment and network analyses, and PCA for temporal analysis.
Main Results:
- Significant gene down-modulation (80%) and reversion to osteoblast-like phenotype.
- Enrichment in TGFbeta signaling pathways (AKT1, SMADs).
- Modulation of cancer-related miRs, including miR-34a, miR-26b, and miR-378.
- miR-34a identified as a key regulator of TGFbeta signaling genes during differentiation.
Conclusions:
- miR-34a expression and TGFbeta signaling down-modulation are critical for CD99-mediated OS cell differentiation.
- Specific molecular players involved in OS differentiation identified.
- Potential therapeutic strategies targeting these actors for differentiation therapy in OS.
