MiR-455-3p suppresses renal fibrosis through repression of ROCK2 expression in diabetic nephropathy

https://doi.org/10.1016/j.bbrc.2018.06.105Get rights and content

Highlights

  • Downregulation of miR-455-3p expression in DN.

  • ROCK2 as a target of miR-455-3p.

  • Re-introduction of ROCK2 attenuated the effect of miR-455-3p on cell proliferation, ECM accumulation and EMT in vitro.

  • Reductions in proinflammatory levels and renal fibrosis by miR-455-3p usages, in vivo.

Abstract

Emerging evidence has shown that microRNAs (miRNAs) play a mediatory role in the pathogenesis of diabetic nephropathy (DN), but the function of the involved miRNAs is still incomplete. Here, we found that miR-455-3p was down-regulated in the human mesangial cells (HMC) and human proximal tubule epithelial cells (HK-2) stimulated with high glucose (HG) or transforming growth factor beta 1 (TGF-β1). Rho-associated coiled coil-containing protein kinase 2 (ROCK2) was identified as a directed target of miR-455-3p. Overexpression of ROCK2 significantly attenuated the inhibitory effects of miR-455-3p on cell proliferation, extracellular matrix (ECM) synthesis and epithelial-mesenchymal transition (EMT) in HG-treated cells. Furthermore, the DN model was prepared by using high-fat feeding combined with Streptozotocin (STZ) induced rats, and the DN group was treated by injecting miR-455-3p agomir. The results of periodic acid-Schiff (PAS) and Masson staining showed that miR-455-3p overexpression improved the pathological changes of glomerular hypertrophy, mesangial amplification, and renal fibrosis. Additionally, miR-455-3p overexpression decreased ROCK2, proliferating cell nuclear antigen (PCNA) and Collagen I levels, and also reduced inflammatory cytokines TNF-α, MCP-1 and IL-1β levels in vivo. Altogether, these results suggest that miR-455-3p plays an essential role in the treatment of renal fibrosis through repressing ROCK2 expression; and miR-455-3p might be an effective therapy for DN.

Introduction

It is well-known that diabetic nephropathy (DN) is a leading cause of end-stage renal disease globally and an independent risk factor for all-cause including cardiovascular mortalities in obesity and diabetic patients [1]. These independent risk factors are structurally characterised by an early thickening of glomerular and tubular basement membranes as a result of the excessive accumulation of the extracellular matrix (ECM) [2]. ECM deposition in the tubulointerstitium and glomerular mesangium has been associated with a progressive decline in renal function of diabetes [2,3]. Tubulointerstitial fibrosis and glomerulosclerosis are related to renal dysfunction [[4], [5], [6]]. Fibrosis is at the core of the high morbidity and mortality rates linked with DN. To date, specific therapeutic options with this target are still not yet available in the clinics [7]. DN is a predominantly substantial risk factor for the development of various macrovascular complications such as atherosclerosis and stroke [8]. Hence, identification of molecular pathways which contribute to the pathophysiology of DN is imperative for the development of new therapeutic strategies.

Nowadays, microRNAs (miRNAs) are under intense exploration as potent regulators of various critical health diseases with a potentially crucial bearing on other disease initiation and progression including diabetic kidney disease [[9], [10], [11]]. miRNAs are small non-coding RNA transcripts with a length of 22 nucleotides and through post-transcriptional binding of the 3′untranslated regions (3′UTR) of miRNA targets, the interactions within themselves may lead to the repression of some genes, associated protein expressions and translational inhibition of protein synthesis [10,11]. A single miRNA can alter the appearance of a large number of target genes, and therefore convincing a specific pathology by regulating signallings and cascades the whole disease-specific pathway [12]. Accumulating studies reported that some miRNAs had been shown to be relevant to fibrotic processes in DN. For instance, miR-21 antagonism in STZ-induced diabetic mice decreased mesangial expansion, interstitial fibrosis, macrophage infiltration, and fibrotic- and inflammatory gene expression [12]. Inhibiting miR-192 ameliorates renal fibrosis in DN [13] and miR-135a promotes renal fibrosis in DN by regulating TRPC1 [11]. Several studies suggested that miR-455-3p may be linked to fibrosis in some critical disease. Ong et al. showed that miR-455-3p was induced by TGF-β1 in lung fibroblasts [14]. Huang et al. found that miR-455 was significantly downregulated in diabetic mouse myocardium and Ang II-induced cardiac fibroblasts [15]. Wu et al. reported that the restoration of miR-455 gene expression might thus be a potential therapeutic strategy to reverse pressure-induced cardiac hypertrophy and prevent maladaptive cardiac remodelling through the regulation of miR-455 at different time points following hypertrophy [16]. More importantly, miR-455-3p was down-regulated in serum from STZ-induced diabetic mice [12]. Based on the above results, we speculated that miR-455-3p might be associated with renal fibrosis in DN.

In this work, we found that miR-455-3p was down-regulated in HMC and HK-2 cells with HG or TGF-β1 stimulation. We further revealed that ROCK2 was a directed target of miR-455-3p and abolished the special effects of miR-455-3p on cell proliferation, and EMT, fibrotic- and proliferative-gene expression in HG-treatment HMC and HK-2 cells. Moreover, we indicated that miR-455-3p overexpression in vivo could dramatically attenuate the increases in parameters of glomerular expansion and renal fibrosis of DN induction in rat. The pharmacological synergy of miR-455-3p and its incorporation into the medicine would be feasible to end the short- and long-term complications of renal fibrosis in DN.

Section snippets

Cell culture and transfection

The primary HMC cell lines were purchased from the ScienCell Corporation (Carlsbad, CA, USA) and were cultured in DMEM media, containing 10% FBS. HK-2 cells were acquired from the Chinese Academy of Sciences (Shanghai branch, China) and were cultured in mixed DMEM with F12 medium (Life Technologies) containing 10% FBS. miR-455-3p mimics and its negative control were purchased from RiboBio (Guangzhou, China). The coding region of the ROCK2 mRNA was amplified by PCR from genomic DNA, and was

Downregulation of miR-455-3p expression in DN

To explore the potential role of miR-455-3p in the HMC and the HK-2, we analysed the expression of miR-455-3p in HMC and HK-2 cells stimulated with HG or TGF-β1. As shown in Fig. 1A and B, miR-455-3p expression was significantly reduced at different time points (0, 12, 24 and 48 h) following stimulation with HG (25 mmol/L) by qRT-PCR. Consistent with the above results, the expression of miR-455-3p was also down-regulated in a time-dependent manner in both HMC and HK-2 cells with TGF-β1

Discussion

In this study, we have learnt that miR-455-3p overexpression could significantly reduce the HMC and HK-2 cell proliferation, cell cycle, and synthesis of Collagen I especially when activated by either HG or TGF-β1. Likewise, our results disclosed that ROCK2 was a target gene of miR-455-3p and the transfection with ROCK2 overexpression could reduce the effects of miR-455-3p on cell proliferation and ECM synthesis. Correspondingly, overexpression of miR-455-3p could significantly attenuate the

Acknowledgments

This study was supported by Chinese and western medicine combined with type I plateau discipline project of Shanghai University of Traditional Chinese Medicine and a Grant-in-Aid from Shanghai Hongkou district 2015–2017 medical and health key specialty construction project.

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