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Fig. 1
Identification, characterization, and conservation of miRNAs targeting HCRT. (A) In silico prediction of HCRT-targeting miRNAs (from the miRNA bodymap). hsa-miR-137, hsa-miR-637, and hsa-miR-654-5p were predicted to target human HCRT. mmu-miR-137-3p and mmu-miR-665-3p were predicted to target Hcrt in mice. (B) Thermodynamic energy prediction for the association of human HCRT 3′-UTR or mouse Hcrt 3′-UTR with the predicted miRNAs. Green: miRNAs; red: Human HCRT/mouse Hcrt sequence. (C) Effect on HCRT levels following overexpression of precursor hsa-miR-137 (n = 5), hsa-miR-637 (n = 6), hsa-miR-654-5p (n = 3), or scrambled miRNA control (n = 8) in the human neuroblastoma cell line SK-N-MC. RNA levels are presented as the magnitude of difference from the control ± SEM; *P < 0.05; **P < 0.01; one-way ANOVA with Dunnett’s post hoc test. The mature miRNA level was quantified in the same cells and are presented as fold change compared to control ± SEM ***P < 0.001, two-tailed Student’s t test for each independent experiment as marked with vertical lines. (D) Direct miRNA-mediated downregulation of the luciferase signal with the 3′-UTR of HCRT cloned into the psicheck-2 luciferase vector. The vector and miRNAs were cotransfected into HEK-293 cells. miR-107 and miR-103 were used as negative controls as these do not target HCRT. Mutation of miR-137-seed site:mut-137, mutation of miR-637-seed site:mut-637, and mutation of miR-654-5p-seed site:mut-654. Data were normalized to the control vector in each experiment ± SEM (n = 5/condition). *P < 0.05; **P < 0.01; ***P < 0.001; four separate one-way ANOVA with Dunnett’s post hoc tests are used compared to the control groups vector, Mut-137 vector, Mut-637 vector, or Mut-654 vector. The four experiments are separated by vertical lines.