MiR-27b-3p inhibits the progression of renal fibrosis via suppressing STAT1

Abstract

Renal fibrosis is a pathologic change in chronic kidney disease (CKD). MicroRNAs (miRNAs) have been shown to play an important role in the development of renal fibrosis. However, the biological role of miR-27b-3p in renal fibrosis remains unclear. Thus, this study aimed to investigate the role of miR-27b-3p in the progression of renal fibrosis. In this study, HK-2 cells were stimulated with transforming growth factor (TGF)-β1 for mimicking fibrosis progression in vitro. The unilateral ureteric obstruction (UUO)-induced mice renal fibrosis in vivo was established as well. The results indicated that the overexpression of miR-27b-3p significantly inhibited epithelial-to-mesenchymal transition (EMT) in TGF-β1-stimulated HK-2 cells, as shown by the decreased expressions of α-SMA, collagen III, Fibronectin and Vimentin. In addition, overexpression of miR-27b-3p markedly decreased TGF-β1-induced apoptosis in HK-2 cells, as evidenced by the decreased levels of Fas, active caspase 8 and active caspase 3. Meanwhile, dual-luciferase assay showed that miR-27b-3p downregulated signal transducers and activators of transcription 1 (STAT1) expression through direct binding with the 3'-UTR of STAT1. Furthermore, overexpression of miR-27b-3p attenuated UUO-induced renal fibrosis via downregulation of STAT1, α-SMA and collagen III. In conclusion, miR-27b-3p overexpression could alleviate renal fibrosis via suppressing STAT1 in vivo and in vitro. Therefore, miR-27b-3p might be a promising therapeutic target for the treatment of renal fibrosis.

Keywords: Chronic kidney disease; Renal fibrosis; Unilateral ureteral obstruction; miR-27b-3p.

Fig. 1

Overexpression of miR-27b-3p attenuated TGF-β1-mediated EMT in HK-2 cells. a HK-2 cells were treated with 5 ng/ml TGF-β1 for 48 h. RT-qPCR was used to measure the level of miR-27b-3p in HK-2 cells. b The level of miR-27b-3p in HK-2 cells transfected with miR-27b-3p agonist was detected using qRT-PCR. c HK-2 cells were transfected with miR-27b-3p agonist for 6 h, and then exposed to 5 ng/ml TGF-β1 for 48 h. Expressions of α-SMA, Collagen III, Fibronectin and Vimentin in HK-2 cells were detected with western blotting. Images are representative of three independent experiments. d–g The relative expressions of α-SMA, Collagen III, Fibronectin and Vimentin in HK-2 cells were quantified via normalization to GAPDH. **p < 0.01, compared with the control group. ^##p < 0.01, compared with the TGF-β1 group

Fig. 2

STAT1 was a binding target of miR-27b-3p. a Sequence alignment of miR-27b-3p with the binding sites within the WT or MT regions of STAT1. b The luciferase activity in HK-2 cells following co-transfecting with STAT1-WT/MT 3′-UTR plasmid and miR-27b-3p agonist were detected using dual-luciferase reporter assay. c HK-2 cells were transfected with miR-27b-3p agonist for 6 h, and then exposed to 5 ng/ml TGF-β1 for 48 h. The level of STAT1 in HK-2 cells was detected by RT-qPCR. **p < 0.01, compared with the control group. ^##p < 0.01, compared with the TGF-β1 group

Fig. 3

Overexpression of miR-27b-3p inhibited TGF-β1-induced apoptosis in HK-2 cells. HK-2 cells were transfected with miR-27b-3p agonist for 6 h, and then exposed to 5 ng/ml TGF-β1 for 48 h. a, b Apoptotic cells were detected with Annexin V and PI double staining. (C) Expressions of p-STAT1, STAT1, Fas, active caspase 8, active caspase 3 in HK-2 cells were detected with western blotting. Images are representative of three independent experiments. d–h The relative expressions of p-STAT1, STAT1, Fas, active caspase 8, active caspase 3 in HK-2 cells were quantified via normalization to GAPDH. **p < 0.01, compared with the Control group. ^##p < 0.01, compared with the TGF-β1 group

Fig. 4

Overexpression of miR-27b-3p alleviated renal fibrosis in vivo. a Analysis of kidney injury in UUO kidneys by H&E staining (magnification, × 200). black arrows pointed to fibroblasts. b Representative photomicrographs of PAS-stained kidney sections (magnification, × 400). Black arrows pointed to glomerular mesangial matrix; red arrows pointed to exfoliation of renal tubular epithelial cells; blue arrows pointed to renal interstitial edema. c Analysis of collagen deposition and renal fibrosis by Masson’s trichrome staining (magnification, × 200). Blue arrow pointed to collagen fibers. d Total lung fibrotic area was measured by Image-Pro Plus. e Integrity of the glomerulotubular junction and proximal tubular mass were determined in LTL staining (magnification, × 200). **p < 0.01, compared with the control group. ^##p < 0.01, compared with the UUO group

Fig. 5

Overexpression of miR-27b-3p alleviated renal fibrosis in vivo. a The level of BUN in urine of mice was detected by ELISA. b The level of CR in urine of mice was detected by ELISA. (C) RT-qPCR analysis showed the level of miR-27b-3p in kidney tissues in UUO mice model. d Expression levels of p-STAT1, STAT1, α-SMA, Collagen III in kidney tissues were detected with western blotting. Images are representative of three independent experiments. e–h The relative expressions of p-STAT1, STAT1, α-SMA, Collagen III in kidney tissues were quantified via normalization to GAPDH. **p < 0.01, compared with the control group. ^##p < 0.01, compared with the UUO group

Fig. 6

Overexpression of miR-27b-3p inhibited TGF-β1-induced apoptosis in HK-2 cells via inhibiting STAT1. a–d Western blot analysis of p-STAT1, STAT1 and active caspase 3 expressions in HK-2 cells treated with 5 ng/ml TGF-β1, STAT1 OE and miR-27b-3p agonist. e, f Apoptotic cells were detected with Annexin V and PI double staining. **p < 0.01