Fig 2: miR-4793-3p negatively regulates the TGF-ß pathway. (A) In TGF-ß-treated THP-1 cells, the expression levels of M2-related makers, CXCR4, IL-10 and ARG1, were significantly upregulated. By contrast, the expression levels of M2-related makers, HLA-DR, IL-12, CD80, iNOS, TNF-a and MCP-1, were significantly downregulated. (B) TGF-ß-treated THP-1 cells increased Grem1, Smad2 and Smad3 levels compared with controls. When the cells were transfected with miR-4793-3p mimics, the expression levels of Grem1, Smad2 and Smad3 significantly decreased. There was no significant change in the expression levels of Grem1, Smad2 or Smad3 in the specific miR-4793-3p mimic NC group compared with controls. *P<0.05; **P< 0.01 and ***P<0.001. CXCR4, C-X-C motif chemokine receptor 4; ARG1, arginase 1; HLA-DR, human leukocyte antigen-DR isotype; iNOS, inducible nitric oxide synthase; MCP1, monocyte chemoattractant protein; NC, negative control; ns, not significant; miR, microRNA.

Fig 3: Grem-1 is a direct target of iBETs. ChIP assays were performed in renal samples using an antibody specific for BRD4 or normal rabbit IgG, the latter being a negative control. Enrichment of BRD4-binding regions in the promoter of mouse Grem-1 was quantified by qPCR using specific primers. In each group, samples were pulled (4–7 mice per group). Data are from two independent experiments and each qPCR was run in triplicate. Results are expressed as the n-fold enrichment of anti-BRD4 antibody relative to the negative control antibody (considered to be 1). * p < 0.05 vs. control; # p < 0.05 vs. NTS-injected mice.

Fig 4: Grem1 is a target gene of miR-4793-3p. (A). Dual-Luciferase assay demonstrated significantly declined luciferase activity in the 293T cells, which were co-transfected with Grem1-WT and miR-4793-3p mimics compared with mimic-NC. By contrast, no significant change in luciferase activity was observed in cells co-transfected with Grem1-MUT and miR-4793-3p mimics compared with mimic-NC. (B) Significantly higher expression of miR-4793 was detected by fluorescent reverse transcription-quantitative PCR in 293T cells transfected with miR-4793-3p mimics compared with cells transfected with miR-4793-3p mimic NC or the control. No significant difference was observed between miR-4793-3p mimic NC and the control groups. (C) In TGF-ß-treated THP-1 cells transfected with miR-4793-3p mimics, significantly downregulated Grem1 mRNA levels was observed compared with the control. By contrast, the mRNA levels of Grem1 was not significantly different between the miR-4793-3p mimic NC group and the control. (D) Western blotting showed significantly downregulated Grem1 protein levels in the miR-4793-3p mimics group compared with the control. There was no significant change in Grem1 protein levels in the miR-4793-3p mimic NC group compared the control. ß-actin served as the internal reference gene. **P<0.01 and ***P<0.001. WT, wild-type; MUT, mutant; NC, negative control; ns, not significant; miR, microRNA; Grem1, Gremlin-1.

Fig 5: JQ1 diminishes Gremlin-1 in an experimental model of progressive tubulointerstitial fibrosis. Unilateral ureteral obstruction (UUO) was performed in C57Bl/6 mice as described in Methods, and kidneys were studied after 5 days. Some mice were treated with JQ1 (100 mg/kg body weight per day, i.p. starting 24 h before UUO). RNA was obtained from total renal extracts and Grem-1 mRNA levels were determined by real-time qPCR. Data are expressed as the mean ± SEM of 5–7 animals per group. * p < 0.05 vs. control; # p < 0.05 vs. UUO.