Fig. 5: Targeting C19MC families with short antisense oligonucleotides.
a, Heatmap showing the overlap of C19MC targets detected in eCLIP with predicted targets from TargetScan. C19MC members are grouped into 3p (top, n = 10) and 5p (bottom, n = 13) miRNAs. The black dots indicate the largest overlap with predicted targets. The number of high-confidence peaks for each member are indicated. b, Mature C19MC miRNAs as in a. The AAGUGC seed motif in 3p miRNAs is highlighted in red. LNAs complemented the seed sequences of one or more C19MC members (7-nt-long, complementary to position 2–8). Nontargeting controls were designed (CTRL 1, CTRL 2). Predicted melting temperatures are indicated. c, Schematic illustration of the miRNA targeting mechanism of LNAs. d, Heatmap showing the confluency of BT183 ETMR cells treated with LNAs over time, tracked using live-cell microscopy in two-dimensional (2D) culture with four biological replicates. P values were calculated with a one-way analysis of variance (ANOVA) and adjusted P values are provided. The results of n = 4 biological replicates are shown. e, Longitudinal doubling time estimation for BT183 growth treated with the indicated LNAs. Doubling times were calculated using the log of exponential growth equation in Prism 8. The results of n = 4 independent experiments are shown. f, Bright-field images showing BT183 cell confluency after treatment with anti-517-3p or CTRL 1 LNAs for 72 h. Identified cell areas are highlighted in red. This experiment achieved reproducible results in four biological replicates. g, Dot plot showing the caspase-3 and caspase-7 signal after 72 h of LNA treatment in three-dimensional BT183 cells. The means are indicated. Statistical analysis was performed on n = 4 biological replicates using a one-way ANOVA, comparing each condition to CTRL 1. Padj values are given. h, Bright-field and fluorescence images showing the caspase-3 and caspase-7 signal after 72 h of treatment with anti-517-3p or CTRL 1 LNAs in BT183 cells. Images shown are representative of n = 4 biological replicates. i, IF image analysis of BT183 cells for Ki-67 and cleaved caspase-3 (CC3). Cells were treated with anti-517-3p or CTRL 1 LNAs for 72 h. This experiment achieved reproducible results in three biological replicates. j, Representative IF staining for the indicated markers. Cells were treated with anti-517-3p or CTRL 1 LNAs for 72 h. This experiment achieved reproducible results in three biological replicates. Left, The dashed boxes indicate the tumor area shown in the magnified panels on the right. f, Scale bars, 400 µm. h, Scale bars, 500 µm. i,j, Scale bars, 300 µm. Schematic in c created with BioRender.com.
