Spherical nucleic acid targeting microRNA-99b enhances intestinal MFG-E8 gene expression and restores enterocyte migration in lipopolysaccharide-induced septic mice.

Xiao Wang,Chad A Mirkin,Yulan Liu,Heng-Fu Bu,Alexander W Scott,Fangyi Liu,Xiao-Di Tan,Liangliang Hao,Ke Tian,Isabelle G De Plaen

doi:10.1038/srep31687

Abstract

Milk fat globule-EGF factor 8 (MFG-E8) maintains the intestinal homeostasis by enhancing enterocyte migration and attenuating inflammation. We previously reported that sepsis is associated with down-regulation of intestinal MFG-E8 and impairment of enterocyte migration. Here, we showed that impairment of intestinal epithelial cell migration occurred in lipopolysaccharide (LPS)-induced septic mice. Treatment of RAW264.7 cells (a murine macrophage-like cell line) with LPS increased expression of miR-99b, a microRNA that is predicted to target mouse MFG-E8 3′UTR. Using a luciferase assay, we showed that miR-99b mimic suppressed the activity of a reporter containing MFG-E8 3′UTR. This suggests the role of miR-99b in inhibition of MFG-E8 gene expression. In addition, we developed an anti-miR99b spherical nucleic acid nanoparticle conjugate (SNA-NCanti-miR99b). Treatment of both naïve and LPS-challenged cells with SNA-NCanti-miR99b enhanced MFG-E8 expression in the cells. Administration of SNA-NCanti-miR99b rescued intestinal MFG-E8 expression in LPS-induced septic mice and attenuated LPS inhibitory effects on intestinal epithelial cell migration along the crypt-villus axis. Collectively, our study suggests that LPS represses MFG-E8 expression and disrupts enterocyte migration via a miR-99b dependent mechanism. Furthermore, this work shows that SNA-NCanti-miR99b is a novel nanoparticle-conjugate capable of rescuing MFG-E8 gene expression and maintaining intestinal epithelial homeostasis in sepsis.

Highlights

Epithelial restitution results in delaying mucosal wound healing
To understand the role of endotoxemia in intestinal epithelial barrier homeostasis, we studied the pathophysiological effect of LPS-induced sepsis on enterocyte migration along crypt-villus axis in mice using in vivo bromodeoxyuridine (BrdU) pulse-chase analysis
We found that the level of luciferase activity in the cells transfected with pSGGmfge8-3′UTR were significant lower than that in cells transfected with empty vector (Fig. 3B), suggesting that the Milk fat globule-EGF factor 8 (MFG-E8) 3′UTR is a functional unit for maintaining homeostasis of MFG-E8 gene expression at a post-transcriptional level

Summary

Introduction

Epithelial restitution results in delaying mucosal wound healing. Currently, effective approach for maintaining intestinal cell migration in critical illnesses is lacking. Evidence further shows that MFG-E8 inhibits tissue fibrosis by promoting the removal of collagens from inflammatory tissues[13]. We previously showed that MFG-E8 promotes intestinal epithelial cell migration in vitro and in vivo[6]. Down-regulation of MFG-E8 contributes to delayed intestinal epithelial cell migration and restitution in inflammation[6,15]. We have studied the 5′-promoter region-associated transcriptional regulation of the MFG-E8 gene expression in physiological and inflammation conditions[18]. In the present study, we further examined the involvement of 3′UTR and miRNAs in suppressing MFG-E8 gene expression in lipopolysaccharide (LPS)-induced sepsis condition. To protect against sepsis-induced impairment of intestinal homeostasis, we developed a novel approach which targets MFG-E8 associated miRNA, rescuing MFG-E8 gene expression, using spherical nucleic acid (SNA) nanoparticle conjugate (NC) technology

Methods

Results

Conclusion