LncRNA H19 accelerates renal fibrosis by negatively regulating the let-7b-5p/TGF-βR1/COL1A1 axis

Abstract

Background: Transforming growth factor-beta1 (TGF-β1)-mediated renal fibrosis is a critical pathological process of chronic kidney disease worsening to end-stage renal disease. Recent studies have shown that long noncoding RNA H19 (lncRNA H19) is widely involved in the formation and progression of fibrosis in multiple organs. However, its molecular events in renal fibrosis remain to be elucidated.

Methods: Rats were treated with adenine intragastrically and HK-2 cells were induced by TGF-β1 to construct renal fibrosis models in vivo and in vitro, respectively. Renal histopathological examination was performed using HE and Masson staining. Gene expression levels of interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), TGF-β1, fibronectin (Fn), alpha-smooth muscle actin (α-SMA), H19, let-7b-5p, TGF-β receptor 1 (TGF-βR1), and type I collagen (COL1A1) were detected by qRT-PCR. Immunohistochemistry, immunofluorescence, and western blot analysis were used to evaluate the expression of renal fibrosis biomarkers. Dual-luciferase reporter assay was used to verify the presence of binding sites between H19 and let-7b-5p, and between let-7b-5p and TGF-βR1 and COL1A1.

Results: H19 was overexpressed in both in vivo and in vitro renal fibrosis models. H19 knockdown significantly reversed TGF-β1-induced upregulation of fibronectin, COL1A1, and α-SMA and downregulation of E-cadherin in HK-2 cells, accompanied by an increase in let-7b-5p. Let-7b-5p was bound to H19 in HK-2 cells, and its overexpression inhibited TGF-β1-induced HK-2 cell fibrosis. Further experiments determined that let-7b-5p directly targets TGF-βR1 and COL1A1 in HK-2 cells. In addition, inhibition of let-7b-5p reversed the reduction in HK-2 cell fibrosis induced by H19 knockdown. Finally, knockdown of H19 alleviated renal fibrosis in vivo and was associated with regulation of the let-7b-5p/TGF-βR1/COL1A1 axis.

Conclusion: Our results indicate that knockdown of H19 inhibits renal tubular epithelial fibrosis by negatively regulating the let-7b-5p/TGF-βR1/COL1A1 axis, which may provide new mechanistic insights into CRF progression.

Keywords: Extracellular matrix; Let-7b-5p; Renal fibrosis; TGF-β1; lncRNA H19.