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. 2024 Sep 21;25(18):10147. doi: 10.3390/ijms251810147

Figure 5.

Figure 5

β-estradiol (E2) regulates the expression of essential metabolic proteins via miR-10a/b-5p. (a) Ingenuity Pathway Analysis of the target genes of miR-10a/b-5p and E2. (b) Seed match target sequences of miR-10a/b-5p in the 3′ UTR of NCOR2 in humans and mice. (c) Schematic illustration of the target validation mechanism and maps of the pLenti-Luc-10a-Ncor2 vectors. In this design, the mouse pre-mir-10a (110 bp) with an artificial intron is strategically inserted within the luciferase (Luc) gene, resulting in separated Luc-a and Luc-b exons. The wild type (WT) or mutant (Mut) target-binding sequence of the mouse Ncor2 is then introduced into the 3′ UTR of the Luc gene. Upon transcription, a miR-10a duplex comprising mature miR-10a-5p and miR-10a-3p strands is generated from the artificial intron, which encodes a pre-mir-10a spliced out from the primary Luc-a and Luc-b transcripts. The miR-10a-5p strand is preferentially selected within the RNA-induced silencing complex (RISC), forming the miRISC that specifically binds to the Ncor2 WT target site (TS), but not the Mut TS. The exonic segments of Luc-a and Luc-b then join to produce functional LUC protein (luciferase), whose activity is downregulated by the targeting action of miR-10a-5p. Moreover, inhibition of miR-10a-5p by its inhibitor mitigates its targeting effect on luciferase. Luc, luciferase gene; pCMV, CMV promoter; WPRE, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element DNA sequence; miRISC, miRNA-RNA-induced silencing complex. (d) Expression of miR-10a-5p in HEK293T cells transfected with the luciferase reporter plasmid containing the murine pre-mir-10a insertion (pLenti-Luc-10a) and without insertion (pLenti-Luc). pLenti-Luc-10a vector was used to insert the NCOR2 wild type target site (WT TS) or the mutant target site (Mut TS) at the end of luciferase gene (c). n = 3 per group. (e) Luciferase activity in HEK293T cells transfected with pLenti-Luc-10a, pLenti-Luc-10a-WT TS, pLenti-Luc-10a-Mut TS, or co-transfected with miR-10b-5p inhibitor (pLenti-Luc-10a-WT TS-Inh). NTC, non-transfected cells. n = 3 per group. (f) Effects of E2 on expression of miR-10a/b-5p in NIT-2 cells. n = 5–6 per group. (g,h) Automated western blots and quantified protein expression of INS, INSR, and NCOR2 in NIT-2 cells cultured in a normal glucose medium (1 mg/L, NG) or a high glucose medium (10 mg/L, HG) and treated with E2 for 24 h. A protein marker (L) with corresponding molecular weights (kDa) is shown. n = 5 per group. * p < 0.05, ** p < 0.01 (versus given NT in a HG). n = 5–6 per group. Error bars indicate the SEM derived from one-way ANOVA.