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. 2021 Feb 3;11:627007. doi: 10.3389/fgene.2020.627007

Figure 1.

Figure 1

Identification of USP18-targeting miRNAs. (A) Prediction of USP18-targeting miRNAs using RNA hybrid algorithm and mirWalk software (TargetScan, Pita, miRDB, and RNA22). The common high-scoring miRNAs (27) were further selected for expression (counts per million mapped reads, CPM > 50) in 90 human cell types (FANTOM5 dataset). The selected candidates (14) are listed. (B) The 14 selected miRNAs and a miR-control (miR-ctrl) were expressed as mimics in HeLa S3 cells and USP18 mRNA was measured by qPCR (n = 4). Results (± SEM) shown as expression (2-ΔCt) relative to 18S, used as housekeeping gene. Raw-matched one-way ANOVA with Geisser-Greenhouse correction was performed (Dunnett’s multiple comparison – compared to miR-ctrl), adjusted values shown as stars *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (C) The miR-ctrl and the indicated miRNAs were expressed as mimics in HeLa S3 cells and 48 h later endogenous USP18 was measured by western blot. Actin B (ACTB) as loading control. The data are representative of three experiments. (D) Schematic of the USP18 mRNA. Numbered boxes correspond to the exons. CDS, coding sequences. Arrowheads, the start (ATG) and stop (TAA) codons and the binding sites for the four indicated miRNAs. Note that exon 11 (in red) contains 43 coding nt, the stop codon, and the 3’untranslated region (UTR). The sequences of USP18 exon11 are provided as Supplementary Information. (E) Binding sites (BS) of miR-191-5p, miR-24-3p, miR-423-5p, and miR-532-3p in the USP18 3’UTR. Seed-matched sequences are in red. Pairing of miRNA-USP18 sequences was obtained with the RNAhybrid algorithm. (F) HeLa S3 cells were transfected with the indicated miRNAs and 24 h later with the psiCHECK2-USP18 3’UTR reporter plasmid, WT, or mutated in the miRNA binding site (BS mut). Each mutant plasmid contains a 5 nt mutation (nt 2–6 of the seed-matched sequence shown in E). Luciferase activity was measured 24 h after plasmid transfection (n = 4) and expressed as Renilla/Firefly ratio (± SEM).