Autoregulatory loop between TGF-β1/miR-411-5p/SPRY4 and MAPK pathway in rhabdomyosarcoma modulates proliferation and differentiation

The origin of rhabdomyosarcoma (RMS) remains controversial. However, specific microRNAs (miRNAs) are downregulated in RMS and it is possible that re-expression of these miRNAs may lead to differentiation. Transforming growth factor-β1 (TGF-β1) is known to block differentiation of RMS. We therefore analyzed miRNA microarrays of RMS cell lines with or without TGF-β1 knockdown and identified a novel anti-oncogene miR-411-5p. Re-expression of miR-411-5p inhibited RMS cell proliferation in vitro and tumorigenicity in vivo. Using a luciferase reporting system and sequence analysis, the potential target of miR-411-5p was identified as sprouty homolog 4 (SPRY4), which inhibits protein kinase Cα-mediated activation of mitogen-activated protein kinases (MAPKs), especially p38MAPK phosphorylation. These results revealed an inverse correlation between TGF-β1/SPRY4 and miR-411-5p levels. SPRY4 small interfering RNA and miR-411-5p both activated p38MAPK phosphorylation and also promoted apoptosis and myogenic differentiation, indicated by increased caspase-3, myosin heavy chain, and myosin expression. SPRY4 and miR-411 mRNA levels correlated with TGF-β1 expression levels in RMS tissues, which was confirmed by immunohistochemical staining for TGF-β1, SPRY4, and phosphorylated p38MAPK proteins. Overall, these results indicate that miR-411-5p acts as an RMS differentiation-inducing miRNA prompting p38MAPK activation via directly downregulating SPRY4. These results establish an autoregulatory loop between TGF-β1/miR-411-5p/SPRY4 and MAPK in RMS, which governs the switch between proliferation and differentiation.