Fig. 3: NR4A2 is a transcriptional activator of miR-203a-3p. | Nature Communications

Fig. 3: NR4A2 is a transcriptional activator of miR-203a-3p.

From: Epigenetic regulation of beta-endorphin synthesis in hypothalamic arcuate nucleus neurons modulates neuropathic pain in a rodent pain model

Fig. 3

a Schematic diagrams of distinct constructs of pGL3 luciferase reporters (pGL3-F1 to F5). b Luciferase reporter assay of the transcriptional activities of pGL3-F1 to F5 as indicated in (a). **p = 0.0015 versus pGL3-F4, two-sided Student’s t test. Data are from six independent experiments. c The JASPAR algorithm predicted that ΔF in the miR-203a-3p promoter region harbors five putative protein binding elements, including SP1, NFKB1, NFATC2, NR4A2, and SOX10. d RT‒PCR analysis of above five transcription factors in the ARC of intact rats as well as in PC12 cells. Samples without reverse transcriptase (-RT) were used as negative controls. Similar results were obtained in four independent experiments and the data shown were from one representative experiment. e Schematic diagrams of the luciferase reporter constructs including mut-SP1, mut-NFKB1, mut-NFATC2, mut-NR4A2, and mut-SOX10. f Luciferase activities of miR-203a-wt, mut-SP1, mut-NFKB1, mut-NFATC2, mut-NR4A2 and mut-SOX10. ***p = 0.0003 versus miR-203a-wt, two-sided Student’s t-test. Data are from six independent experiments. g Immunoblot analysis of the protein abundance of NR4A2 in the ARC. n = 8 rats per group. h FISH analysis of miR-203a-3p (red) combined with immunostaining for NR4A2 protein (green) and DAPI (blue) in the rat ARC on Day 14 following sham or CCI-ION operation. Bar charts show the percentage of both miR-203a+ and NR4A2+ ARC neurons among total numbers of DAPI-labeled cells. *p = 0.0116 versus sham, two-sided Student’s t-test. n = 4 rats per group. Arrows show colocalization. Scale bar, 25 μm. i ChIP‒qPCR analysis indicating the binding of NR4A2 to the miR-203a-3p gene promoter on Day 14 following sham or CCI-ION operation. The fold enrichment of ChIP data was normalized to the input DNA and then compared to the sham group. Data are from three independent experiments. **p = 0.0025 versus sham, two-sided Student’s t-test. n = 6 rats per group. All data are presented as mean values ± SEM.

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