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. 2019 Sep 3;28(1):142–156. doi: 10.1016/j.ymthe.2019.08.014

Figure 8.

Figure 8

miR-92a Reduces Sp1 mRNA and Protein by Directly Targeting the 3′UTR of Sp1, whereas miR-27a Suppresses Sp1 Protein but Not mRNA

(A and B) C57 ESCs were transfected with 1 μg pre-miR-27a or pre-miR-92a miR precursors for 2 days under normoxia, and western blotting and real-time PCR were performed. (A) Sp1 western blotting after overexpression of pre-miR-27a or pre-miR-92a miR precursors. Sp1 protein was reduced by both miRNAs (n = 3; **p < 0.01, ***p < 0.001). (B) Suppression of Sp1 mRNA only by pre-miR-92a but not by pre-miR-27a overexpression in C57 cells (n = 4; **p < 0.01, ***p < 0.001). (C) Sequence alignment of putative miR-27a- and miR-92a-targeting sites within the 3′ UTR of Sp1 (5,331 bp). Two luciferase reporter constructs are shown: fragment A, first half of the 3′ UTR of Sp1 (1–2,760 bp); fragment B, last half of the 3′ UTR of Sp1 (2,321–5,331 bp). (D) Luciferase activity reflecting Sp1 expression was suppressed by pre-miR-92a but not by both pre-miR-27a and pre-miR-NC (n = 6; ***p < 0.001). Sp1-3′ UTR-A-Luc, luciferase reporter containing fragment A of the 3′ UTR of the Sp1 gene; Sp1-3′ UTR-B-Luc, luciferase reporter containing fragment B of the 3′ UTR of the Sp1 gene. Differentiating C57 ESCs were co-transfected with various combinations of 1 μg luciferase reporter, 1 μg pre-miR-27a, 1 μg pre-miR-92a, and 1 μg pre-miR negative control, and luciferase activity was measured 24 h later. (E and F) miR-92a was found to directly target the 3′ UTR of the Sp1 gene. (E) Schematic representation of luciferase reporter constructs showing the predicted structures of each base-paired WT (Sp1 3′ UTR-WT) or mutant (Sp1 3′ UTR-mt1, Sp1 3′ UTR-mt2) fragment B of the Sp1 3′ UTR. (F) Luciferase activity showed the reduction of Sp1 expression by pre-miR-92a and that this suppressive activity of pre-miR-92a was abrogated when the first target site (3,737–3,765 bp) of the 3′ UTR was mutated (n = 6; ***p < 0.001, ###p < 0.001). C57 ESCs were co-transfected with various combinations of 1 μg WT, 1 μg mutant Sp1 3′ UTR, 1 μg pre-miR-92a, and 1 μg pre-miR-NC, and luciferase activity was measured 24 h later. (G) Sp1 was increased even under normoxic conditions. C57 mESCs were transfected for 24 h with an antagomiR against miR-92a (50 nM). Hypoxia: 16 h (n = 3; **p < 0.01, ***p < 0.001). (H) Proposed model for stimulation of the myogenic differentiation of mESCs through the miR-92a/Sp1/MyoD axis.