Upregulation of miR181a/miR212 Improves Myogenic Commitment in Murine Fusion-Negative Rhabdomyosarcoma

Abstract

Fusion-negative rhabdomyosarcoma (FN-RMS) is the most common soft tissue sarcoma of childhood arising from undifferentiated skeletal muscle cells from uncertain origin. Currently used therapies are poorly tumor-specific and fail to tackle the molecular machinery underlying the tumorigenicity and uncontrolled proliferation of FN-RMS. We and other groups recently found that microRNAs (miRNA) network contributes to myogenic epigenetic memory and can influence pluripotent stem cell commitments. Here, we used the previously identified promyogenic miRNAs and tailored it to the murine FN-RMS. Subsequently, we addressed the effects of miRNAs in vivo by performing syngeneic transplant of pre-treated FN-RMS cell line in C57Bl/6 mice. miRNA pre-treatment affects murine FN-RMS cell proliferation in vivo as showed by bioluminescence imaging analysis, resulting in better muscle performances as highlighted by treadmill exhaustion tests. In conclusion, in our study we identified a novel miRNA combination tackling the anti-myogenic features of FN-RMS by reducing proliferation and described novel antitumorigenic therapeutic targets that can be further explored for future pre-clinical applications.

Keywords: microRNA; murine model; pediatric cancer; promyogenic cocktail; promyogenic signaling; rhabdomyosarcoma; skeletal muscle.

FIGURE 1

Identification of differentially expressed genes in murine FN-RMS cell lines. (A) Principal component of murine satellite cells (n = 3) and FN-RMS cell lines (n = 2), with PC1 accounting for 98% of the variance observed in the analysis. (B) Sample distance matrix showing closed proximity of the replicates. (C) Volcano plot of the up- and downregulated genes in murine FN-RMS compared to satellite cells. (D) Selection of Gene Ontology Biological Process pathways up- and down-regulated in murine FN-RMS compared to satellite cells.

FIGURE 2

Selection of previously identified PMC improves myogenic potential in murine FN-RMS. Heatmaps of a selection of genes previously shown to be differentially expressed in MiPs upon PMC treatment (A) analyzed by RNA-seq and (B) confirmed in murine FN-RMS by qRT-PCR using s-PMC. (C) Myogenin expression in green and (D) quantification upon s-PMC treatment in FN-RMS cell lines compared to vehicle. Nuclei are counterstained in blue with HOECHST. s-PMC, Selected Promyogenic Cocktail. Scale bar, 100 μm. *p < 0.05. (E) Reductionist approach to identify the minimal combination of miRNAs from the s-PMC required to have the promyogenic effect. s-AMC, Selected Antimyogenic Cocktail. **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 3

miR-181a/212 target differentiation, proliferation, and migration in murine FN-RMS. (A) Overlap and (B) selection of Gene Ontology Biological Process pathways upregulated in murine FN-RMS and targeted by miR-181a/212. (C) miR-181a/212 increases the myogenic commitment of murine FN-RMS as seen by the quantification of fusion index of MyHC+ myotubes. Nuclei are counterstained in blue with HOECHST. Scale bar, 100 μm. (D) Reduced migration capacity of murine FN-RMS upon miR-181a/212 treatment compared to vehicle. *p < 0.05. (E) Qualitative and quantitative effect of miR-181a/212 on murine FN-RMS proliferation in co-culture setting with human MABs compared to vehicle. Human nuclei were identified with anti-lamin A/C Abs in green. Nuclei are counterstained with HOECHST in blue. Scale bar, 100 μm. **p < 0.01; ***p < 0.001.

FIGURE 4

miR181a/miR212 effects on murine FN-RMS proliferative capacity in vivo. (A) Representative bioluminescent images of C57/Bl6 mice injected in the femoral artery with saline (control) or 1 × 10⁵ KMR46 Fluc+ cells (untreated group) or 1 × 10⁵ KMR46 Fluc+ cells pretreated for 3 days with miR-181a/212 (treated group). Images were taken 7-, 9-, 11-, and 13-days post-injection. (B) Quantification of the BLI signal (n = 3). (C) Functional test by treadmill exhaustion test at 7-, 9-, 11-, and 15-days post-injection. From left to right and top to bottom, graphs of work (J), power (W), distance (m), and time of run (min). (n = 3). **p < 0.01; ***p < 0.001; ****p < 0.0001.