The Function And Mechanism Of MiR-214during Myogenic Differentiation And Rhabdomyosarcoma Development

Vertebrate muscle differentiation is coordinated by an intricate network of transcription factors requiring proliferating myogenic precursors to withdraw irreversibly from the cell cycle. Recent studies have implicated a large number of microRNAs exerting another layer of control in many aspects of muscle differentiation. By annealing to short recognition sequences in the3’-untranslated region, microRNAs attenuate target gene expression through translation repression or mRNA degradation. Here, we show that miR-214promotes myogenic differentiation in mouse C2C12myoblasts at a step preceding the induction of p21and myogenin. Blocking miR-214function with a2’-O-methylated double-stranded inhibitor maintained C2C12cells in the active cell cycle, thereby inhibiting the myogenic differentiation. By global gene expression profiling, we identified the proto-oncogene N-ras as one of miR-214targets. Furthermore, manipulating the N-Ras level with small interfering RNA or adenovirus-mediated forced expression either augmented or attenuated the effect of miR-214, respectively. Thus, our data uncovered a novel microRNA-mediated mechanism that controls myogenic differentiation.Soft tissue sarcomas are a heterogeneous group of mesenchymal tumors that carries a guarded prognosis due to the aggressive local invasion and metastatic potential of these tumors. Progress in the search for etiology and treatment of soft tissue sarcomas is hampered by the fact that they represent a small proportion of all malignancies. Rhabdomyosarcoma (RMS) is a distinct soft tissue sarcoma that likely originates from cells of the myogenic lineage. On the basis of histology, two main subgroups are often described:alveolar and embryonal RMS. Alveolar RMS consists of small, round, and densely packed cells and occurs mainly in the trunk and extremities and carries with it amore unfavorable prognosis. Embryonal RMS typically consists of spindle-shaped cells and occurs mainly in the head and neck region. Until now, treatment of RMS has largely been based on local regional control and toxic systemic chemotherapy regimens without directed cellular therapy. Recent investigations, however, are improving our understanding of its tumor biology and are helping to identify novel prognostic factors and targets for clinical therapy. Our lab has found and proved that miR-214can promote myogenic differentiation by facilitating exit from cell cycle through down-regulation of pro-oncogene Nras. In addition, miR-214is down-regulated in the rhabdomyosarcoma cell lines comparing with human normal skeletal muscles. Therefore, we argue that miR-214may play a tumor suppressor role in the development of rhabdomyosarcoma. To investigate this possibility, we transfected RD cells with ns or214mi, and the cells were subjected to MTT assay, cell count assay and EdU incorporation assay.The result showed that overexpression of miR-214can inhibit the proliferation of RD cell. Moreover, cell cycle analysis showed that re-expression of miR-214can induce G1phase arrest and cell apoptosis. Western blot showed that CDK2was also down-regulated by miR-214. Through immunofluorescence, we also proved that miR-214can promote differentiation of RD cells.Bioinformatics analysis shows that there is only one binding site in the3’-UTR of human Nras,however, western showed that miR-214can down-regulate the Nras protein level. From the above results, we can conclude that miR-214can inhibit the development of RD cells.In order to further study the in vivo roles of miR-214, we generated miR-214conditional knockout mice by inserting two loxP sites flanking the precursor of miR-214. After we got the miR-214fl/+mice, we crossed the miR-214fl/+mice with E2a-cre mice to get miR-214knockout mice. The miR-214-/-mice are viable and fertile. In order to investigate the role of miR-214in muscle regeneration and heart hypertrophy, the wild type and miR-214-/-mice were subjected to cardiotoxin-induced muscle injure and transaortic constriction (TAC) induced heart hypertrophy. Ptchl is a tumor suppressor and ptchl knockout mice exhibit a high incidence of rhabdomyosarcoma RMS. P53+/-mice also showed the development of rhabdomyosarcoma. So we crossed the miR-214-/-mice with Ptchl+/-or P53+/- mice to get the double mutant miR-214-/-;Ptchl+/-or miR-214-/-;P53+/-mice. Then we will analyze the incidence of RMS in these mice and to see whether miR-214knockout will increase the incidence of RMS in Ptchl mutant or P53mutant mice.