RNA immunoprecipitation (RIP)-Chip analysis for EWS-bound mRNA

Although EWS/FLI-1 fusion protein is responsible for most Ewings sarcoma family tumors (ESFT), the function of native EWS remains largely unknown. Here, we first showed that EWS repressed protein expression in a tethering assay. mRNAs bound to EWS were determined by RNA-immunoprecipitation Chip assay, and one of them, proline-rich Akt substrate of 40 kDa (PRAS40) mRNA, directly interacted with EWS. The inhibitor of AKT, API-2, repressed ESFT cell proliferation. We demonstrate that EWS negatively regulated PRAS40 protein expression through binding to PRAS40 3UTR. Furthermore, PRAS40 knockdown inhibited the proliferation and metastatic potential of ESFT cells.

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Huang L, Nakai Y, Kuwahara I, Matsumoto K