MicroRNA-206 expression levels correlate with clinical behaviour of rhabdomyosarcomas

Abstract

Background: Rhabdomyosarcomas (RMSs) are primarily paediatric sarcomas that resemble developing skeletal muscle. Our aim was to determine the effects of microRNAs (miRNA) that have been implicated in muscle development on the clinical behaviour of RMSs.

Methods: Expression levels of miR-1, miR-206, miR-133a and miR-133b were quantified by RT-PCR in 163 primary paediatric RMSs, plus control tissues, and correlated with clinico-pathological features. Correlations with parallel gene expression profiling data for 84 samples were used to identify pathways associated with miR-206. Synthetic miR-206 was transfected into RMS cell lines and phenotypic responses assessed.

Results: Muscle-specific miRNAs levels were lower in RMSs compared with skeletal muscle but generally higher than in other normal tissues. Low miR-206 expression correlated with poor overall survival and was an independent predictor of shorter survival in metastatic embryonal and alveolar cases without PAX3/7-FOXO1 fusion genes. Low miR-206 expression also significantly correlated with high SIOP stage and the presence of metastases at diagnosis. High miR-206 expression strongly correlated with genes linked to muscle differentiation and low expression was associated with genes linked to MAPkinase and NFKappaB pathway activation. Increasing miR-206 expression in cell lines inhibited cell growth and migration and induced apoptosis that was associated with myogenic differentiation in some, but not all, cell lines.

Conclusion: miR-206 contributes to the clinical behaviour of RMSs and the pleiotropic effects of miR-206 supports therapeutic potential.

Figure 1

Box and whiskers plots representing the expression of (A) miR-206, (B) miR-1, (C) miR133a and (D) miR133b in 33 ARMS fusion negative (ARMS_Neg), 45 ARMS PAX3-FOXO1A (ARMS_P3F), 12 ARMS PAX7-FOXO1A (ARMS_P7F), 66 ERMS, 7 RMS not otherwise specified (RMS_NOS), 4 RMS cell lines, 15 normal skeletal muscles (Sk.muscle), 1 myoblasts sample (Myob) and 4 normal tissues. Δ_CT values were calculated by subtracting miRNA CT values from the average CT values of two endogenous controls (RNU6B and RNU48).

Figure 2

Kaplan–Meier plots for overall survival with (A) miR-206 expression (B) miR-1 expression in all RMS samples and (C) miR-206 within fusion gene-negative patients and (D) miR-206 within fusion gene-positive patients. Expression levels within the first quartile was considered ‘low’, ‘med’ when expression was between the second and third quartile and ‘high’ when within the top quartile. P-values were obtained using log-rank test.

Figure 3

Overexpression of miR-206 in RMS cells reduces cell proliferation, cell cycle progression and migration and enhances apoptosis: (A) proliferation and cell viability of RMS cells, (B) cell cycle delay in G₀G₁, (C) apoptosis in all but RH30 cells and (D) reduction in cell migration ^***P<0.001 and ^**P<0.01.