Figure 4: miRNA profile in cell line models expressing E1147K Drosha.
From: Recurrent somatic mutation in DROSHA induces microRNA profile changes in Wilms tumour
(a) Time course experiment of miRNA expression in HEK293 cells transiently expressing wild-type or E1147K Drosha. A schematic representation of the experiment is depicted in the top panel: transfections were performed twice in a 72-h interval (1st trans and 2nd trans), and miRNA expression levels were measured at three time points using the TaqMan Array platform (T1, T2 and T3). The middle panel represents the Short Time-series Expression Miner (STEM) analysis profiles, which were used to cluster and analyse the expression data. Of the six considered profiles produced by STEM analysis (Supplementary Fig. 6), E1147K-transfected cells presented two statistically significant profiles—(profile 1: P=2 × 10−16; and profile 2: P=2 × 10−8) that harboured more genes than expected by chance (48 and 54 genes, respectively). These two profiles represent a reduction in mature miRNA levels from time points T1 to T3. ****P≤0.00001. NS, not significant. (b) miRNA expression in HEK293T cells stably transfected with wild-type or E1147K Drosha. The top panel depicts a schematic representation of the experiment, showing the time length of selection and the passages at which cell extracts were collected for miRNA expression analysis. Four passages of each stable cell line were evaluated for miRNA expression using the TaqMan Array platform, and the mean expression values of each miRNA in E1147K and wild-type cells were compared. The middle panel presents the cDNA sequencing traces, which demonstrate that stable transfection resulted in similar expression levels of the mutant and endogenous wild-type alleles. The lower panel displays a volcano plot showing a trend to preferential reduction in mature miRNAs in E1147K-stably transfected cells, characterized by the enrichment of miRNAs species in the left side of the volcano plot. The x axis represents the log2 fold change between HEK293T-E1147K and wild-type cells; the y axis represents the statistical significance (−log10 P-values).
