Medulloblastoma associated DDX3X mutants restore malignant potential of medulloblastoma cells decreased upon the loss of DDX3X expression

DDX3X is an ATP-dependent RNA helicase. Missense mutations in DDX3X gene are known to occur in WNT, SHH subgroup medulloblastomas. The role of DDX3X in medulloblastoma biology was studied by downregulating its expression in a SHH subgroup Daoy medulloblastoma cell line. DDX3X knockdown resulted in considerable reduction in proliferation, clonogenic potential and anchorage-independent growth of the medulloblastoma cells. Transcriptome analysis was performed to delineate the molecular mechanism underlying reduction in the malignant potential of the medulloblastoma cells upon DDX3X knockdown. Exogenous expression of three DDX3X missense mutants in the DDX3X knockdown cells could restore the malignant potential of the medulloblastoma cells. Overall design: The expression of DDX3X was downregulated in a doxycycline-inducible manner in Daoy medulloblastoma cell line using two independent shRNAs cloned in the lentiviral vector tet-pLKO-puro. One shRNA targets the open reading frame while the other targets 3'-untranslated region (3'-UTR) of the DDX3X mRNA. Daoy cells stably expressing shRNA targeting 3'-UTR of the DDX3X mRNA, were stably transfected witha FLAG-tagged wild type DDX3X or mutant DDX3X cloned in the pCMV10-3xFlag vector. Total RNA was extracted from the parental, vector control, DDX3X shRNA expressing Daoy cells as well those expressing wild type or mutant DDX3X wherein expression of endogenous DDX3X was knocked down. Libraries were prepared using the polyA-enriched RNA fraction of the cells using the Illumina Truseq RNA library preparation kit and sequenced on the Illumina Hiseq 2500 or HiseqX platform.