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. 2021 Jun 2;24(2):547. doi: 10.3892/mmr.2021.12186

Figure 2.

Figure 2.

ANRIL upregulates SIRT1 expression by sponging miR-181a. (A) Putative binding sites (red) between miR-181a and the 3′-UTR of ANRIL and SIRT1. Mutant sequences (blue) of ANRIL were depicted at the top. (B) RNA-binding protein immunoprecipitation assay was conducted to detect the enrichment of miR-181a and ANRIL by using Ago2 antibody to precipitate the lysates of H9c2 cells. Isotype IgG antibody was used as a control. Results are expressed as relative to IgG control (n=3). (C) 293T cells were co-transfected with NC-mimic or miR-181a mimic along with wt or mut ANRIL luciferase reporter plasmids. Luciferase activity was measured at 2 days after transfection. Results are expressed as relative to NC transfection (n=5). (D) H9c2 cells were transfected with pcDNA3.1-vector or pcDNA3.1-ANRIL plasmids, or si-NC or siANRIL as indicated. ANRIL (left) and miR-181a (right) expression levels were determined at 2 days after transfection. (E) 293T cells were co-transfected with NC-mimic or miR-181a mimic along with wt or mut SIRT1 luciferase reporter plasmids. Luciferase activity was measured at 2 days after transfection. Results are expressed as relative to NC transfection (n=5). (F and G) H9c2 cells were transfected with NC-mimic or miR-181a mimic, or antagomir-NC or antagomir-181a. At 2 days after transfection, the (F) mRNA expression levels of miR-181a and SIRT1, and the (G) protein expression levels of SIRT1 were determined (n=3). (H) H9c2 cells were transfected with pcDNA3.1-vector or pcDNA3.1-ANRIL plasmids (4 µg per well) along with or without 50 or 150 nM miR-181a mimic. At 2 days after transfection, cells were cultured under normoxia or exposed to H/R (4 h hypoxia followed by 8 h reoxygenation). SIRT1 protein expression was determined by an immunoblotting assay (n=3). Data are presented as the mean ± SD. **P<0.01. NS, not significant; ANRIL, antisense non-coding RNA in the INK4 locus; SIRT1, sirtuin 1; miR, microRNA; UTR, untranslated region; NC, negative control; wt, wild-type; mut, mutant; si/siRNA, small interfering RNA; H/R, hypoxia/reoxygenation.