Fig. 6

Downregulation of miR-204 target genes upon its expression in medulloblastoma cells. a. Heat map shows top 60 rank ordered genes (GSEA) whose expression is downregulated upon miR-204 expression in the two polyclonal populations, as compared to the doxycycline treated parental HD-MB03 cells and the vector control cells, identified by the RNA-seq analysis. Known validated miR-204 targets M6PR and RAB22A are indicated by arrows. b. GSEA analysis of the genes downregulated upon miR-204 expression in HD-MB03 cells showed most significant enrichment of the Epithelial Mesenchymal transition genes and the ‘miR-204 target gene set’ (software.broadinstitute.org/gsea/msigdb/collections.jsp). c. Real time RT-PCR analysis showing downregulation of the known miR-204 targets RAB22A, M6PR upon miR-204 expression in the medulloblastoma cell lines. d. Y axis shows luciferase activity of the indicated p-Luc construct upon co-transfection with miR-204 expressing plasmid relative to that obtained upon cotransfection with the Vector control. 3′-UTR of the EZR gene and a sponge construct containing six miR-204 binding sites were used as positive controls. e Nucleotide sequence of the miR-204 target site in the 3’UTR of the IGF2R gene and the mutations introduced indicated by ‘$’. f. Western blot analysis showing downregulation of IGF2R protein levels upon miR-204 expression in the polyclonal populations P1, P2 of the indicated medulloblastoma cell line as compared to the doxycycline treated vector control (VC) cells. The numbers below the western blot indicate fold change in the IGF2R protein levels upon miR-204 expression level using GAPDH levels as a house-keeping control for normalization. **, *** indicate p ≤ 0.001 and p ≤ .0001 respectively