Fig 1: Regulation of pro-fibrotic factors by miR-212. (A) HIF1AN siRNA and HIF1AN overexpression plasmid were successfully transfected into cells. (B and C) Expression of a-SMA and HIF-1a. *P<0.05 and **P<0.01 vs. NC. NC, negative control; miR, microRNA; a-SMA, a smooth muscle actin; HIF-1a, hypoxia-inducible factor 1-a; siRNA, small interfering RNA; HIF1AN, hypoxia-inducible factor 1-a inhibitor.
Fig 2: Schematic diagram of the proposed molecular mechanisms of miR-212-mediated renal damage. miR, microRNA; HIF1AN, hypoxia-inducible factor 1-a inhibitor; Ang II, Angiotensin II; COL1A1, collagen a-1(I) chain; COL3A1, collagen a-1(III) chain; a-SMA, a smooth muscle actin; CTGF, connective tissue growth factor.
Fig 3: Regulation of pro-fibrotic factors by miR-212. (A and B) Expression of CTGF and HIF-1a. **P<0.01 vs. NC. NC, negative control; miR, microRNA; CTGF, connective tissue growth factor; HIF-1a, hypoxia-inducible factor 1-a; siRNA, small interfering RNA; HIF1AN, hypoxia-inducible factor 1-a inhibitor.
Fig 4: miR-212 negatively regulates the HIF1AN gene. (A) TargetScan was used to predict HIF1AN as a potential target gene for miR-212. (B) Luciferase activity was detected using Dual-luciferase reporter gene assays. (C and D) Expression of HIF1AN mRNA was detected by reverse transcription-quantitative PCR. (E and F) Protein expression of HIF1AN was detected by western blotting. *P<0.05 and **P<0.01 vs. NC. NC, negative control; miR, microRNA; HIF1AN, hypoxia-inducible factor 1-a inhibitor; wt, wild-type; Mut, mutant.
Fig 5: FIH-1 is a downstream target of miR-122-5p in DN. BUMPT cells were transfected with 200 nM miR-122-5p mimic or negative control (NC) for 24 h and then treated with high glucose (35 mM) for another 24 h. Control cells were maintained in normal medium. (A) The predicted miR-122-5p binding site in 3’UTR of FIH-1 mRNA; (B) immunofluorescence analysis showing the repressive effect of miR-122-5p on FIH-1 expression. Images were collected by laser scanned confocal microscopy, scale bar: 50 µm; (C) immunoblot analysis showing the repressive effect of miR-122-5p on FIH-1 expression. GAPDH was used as internal control. (D) Quantification analysis of the related band intensity. Values are expressed as mean ± SD (n = 4), *p < 0.05. (E) MicroRNA target reporter assay of FIH-1 3'-UTR. The putative miR-122-5p target sequence of the FIH-1 3'-UTR was cloned into the pMIR-REPORT vector. This and empty vector were transfected with miR-122-5p mimic or NC oligonucleotides to analyze luciferase activity. Values are expressed as Mean ± SD (n = 4), *p < 0.05.
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