Downregulation of CRX, a Group 3 specific oncogenic transcription factor, inhibits TGF-b/activin signaling in medulloblastoma cells

Retina-specific gene expression is the distinguishing characteristic of Group 3 medulloblastoma. CRX, a homeobox transcription factor, is overexpressed specifically in Group 3 tumors. ShRNA-mediated CRX knockdown decreased the expression of seveal retina-specific genes and Group 3 specific genes. CRX knockdown inhibited the TGF-beta/activin signaling pathway, which is known to play oncogenic role in a subset of Group 3 medulloblastoma Overall design: Expression of CRX was knocked down in D425 and HD-MB03, Group 3 medulloblastoma cell lines using two independent shRNAs using the Tet-pLKO-puro lentiviral vector. Stable polyclonal populations expressing the shRNA were obtained by transducing the cell lines with the lentiviral particles. Total RNA was prepared from the vector control cells expressing the empty vector and those expressing the shRNA targeting CRX. Libraries were prepared from the polyA-enriched RNA fraction using the Illumina Truseq RNA library preparation kit and sequenced on the Illumina Hiseq platform