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• Research Paper Volume 11, Issue 3 pp 908-920

  Genetical modification on adipose-derived stem cells facilitates facial nerve regeneration

  Relevance score: 16.895367

  Jian Tan, Yipin Xu, Fang Han, Xinhai Ye

  Keywords: facial nerve regeneration, adipose-derived stem cells (ASCs), PLOD1, miR-449

  Published in Aging on February 6, 2019

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  Adipose-derived stem cells (ASCs) have a demonstrative therapeutic potential in aging-associated facial nerve regeneration, in which ASCs work as a source of Schwann cells therapy as an alternative to autologous nerve grafts. However, the transplantation of ASCs may induce local fibrosis, which causes inferior outcome. Here, we aimed to use genetic modification approaches to reduce the fibrogenic properties of ASCs to improve their therapeutic effects on facial nerve regeneration. Since procollagen-lysine 1, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) is essential for hydroxylation of lysine residues in collagen telopeptides and for collagen pyridinoline cross-link formation during fibrosis, and since we found that ASCs expressed high levels of PLOD1, we depleted PLOD1 in ASCs by expression of either a short-hair interfering RNA for PLOD1 (shPLOD1) or a microRNA-449 (miR-449), the latter of which targets PLOD1 mRNA to suppress protein translation. Transplantation of either ASCs-shPLOD1 or ASCs-miR-449 or ASCs-control to repair a 6mm-gap in rat facial nerve was compared. Either ASCs-shPLOD1 or ASCs-miR-449 exhibited a better facial nerve function. Mechanistically, ASCs-shPLOD1 or ASCs-miR-449 significantly and similarly reduced the fibrosis in the injured region, likely through suppression of reactive oxygen species (ROS) and activation of myofibroblasts.

  

  MiR-449 is a PLOD1-targetting miRNA low expressed in ASCs. (A) PLOD1 protein levels were examined in rat and human ASCs by Western blotting. Rat2 and HSFs cells treated with/without TGFβ1 were used as controls. (B) Bioinformatics tools were used to find 3 PLOD1-targeting miRNAs (miR-34, miR-140 and miR-449) conserved in rat and human. (C) RT-qPCR for the 3 PLOD1-targeting miRNAs (miR-34, miR-140 and miR-449) in rat and human ASCs. (D-E) The binding sites of miR-449 on human (D) and rat (E) PLOD1 3’-UTR were shown. *p<0.05. NS: non-significant. N=5.

  
  
  

  

  MiR-449 targets 3’-UTR of PLOD1 mRNA to inhibit its protein translation in ASCs. (A) Rat ASCs cells were transfected with plasmids carrying miR-449 or as-miR-449 or scr as a control. RT-qPCR for miR-449 was done. (B) The intact 3'-UTR of wildtype PLOD1 mRNA (wt PLOD1 3'-UTR) and the 3'-UTR of PLOD1 mRNA with a mutant at miR-449-binding site (mut PLOD1 3'-UTR) were respectively cloned into luciferase reporter plasmids. Rat ASCs cells were then co-transfected with one plasmid from miR-449/as-miR-449/null plasmids and one plasmid from either wt PLOD1 3'-UTR or mut PLOD1 3'-UTR, and subsequently subjected to a dual luciferase reporter assay. (C) RT-qPCR for PLOD1 mRNA in miR-449-modified rat ASCs. (D) Western blotting for PLOD1 protein in miR-449-modified rat ASCs. *p<0.05. NS: non-significant. N=5.

  
  
  

  

  Preparation of ASCs-miR-449 and ASCs-shPLOD1. (A) Schematic of AAVs. 1. an AAV carrying short-hairpin small interfering RNA for PLOD1 (shPLOD1) and a GFP reporter under control of a CMV promoter. 2. an AAV carrying miR-449 and a GFP reporter under control of a CMV promoter. 3. an AAV carrying GFP and scr. (B) Transduced rat ASCs were purified based on GFP expression by flow cytometry. (C) RT-qPCR for PLOD1 levels in ASCs-miR-449, ASCs-shPLOD1 and control ASCs-scr. (D) Western blotting for PLOD1 levels in ASCs-miR-449, ASCs-shPLOD1 and control ASCs-scr. (E) Oil red O staining to evaluate adipogenic induction. (F) Alcian blue staining to evaluate chondrogenetic induction. (G) Von Kossa staining to evaluate osteogenic induction. *p<0.05. NS: non-significant. N=5. Scale bars are 50µm.

  
  
  

  

  Transplantation of either ASCs-miR-449 or ASCs-shPLOD1 exerts better functional facial nerve regeneration than ASCs in rats. Transplantation of either ASCs-shPLOD1 or ASCs-miR-449 or ASCs-control to repair a 6mm-gap in rat facial nerve was compared. The rats were kept for 8 weeks after surgery. (A) The measurement of vibrissae movement grade in 8 weeks after surgery. (B-D) CAMPs analysis at 8 weeks, showing amplitude (B), duration (C) and latency (D) values. (E-F) The number of the myelinated fibers, shown by representative images (E), and by quantification (F). *p<0.05. NS: non-significant. N=10. Scale bars are 50µm.

  
  
  

  

  Transplantation of either ASCs-miR-449 or ASCs-shPLOD1 reduces levels of fibrosis than ASCs during facial nerve regeneration in rats. (A-B) The effects of transplantation of ASCs-miR-449, ASCs-shPLOD1 and control ASCs-scr on the fibrosis were evaluated by Masson-trichrome staining 8 weeks after the surgery in rats, shown by representative images (A), and quantification (B). *p<0.05. NS: non-significant. N=10. Scale bars are 50µm.

  
  
  

  

  Mechanisms underlying the improved anti-fibrotic potential by PLOD1 suppression in ASCs. (A) Western blotting for ROS. (B) DHE assay. (C-F) Western blotting for TGFβ1 (C), Collagen I (D), α-SMA (E) and fibronectin (F), at the site of the skin injury. *p<0.05. NS: non-significant. N=10.