Human umbilical cord mesenchymal stem cell-derived exosomal miR-27b attenuates subretinal fibrosis via suppressing epithelial-mesenchymal transition by targeting HOXC6

Abstract

Background and aim: Subretinal fibrosis resulting from neovascular age-related macular degeneration (nAMD) is one of the major causes of serious and irreversible vision loss worldwide, and no definite and effective treatment exists currently. Retinal pigmented epithelium (RPE) cells are crucial in maintaining the visual function of normal eyes and its epithelial-mesenchymal transition (EMT) is associated with the pathogenesis of subretinal fibrosis. Stem cell-derived exosomes have been reported to play a crucial role in tissue fibrosis by transferring their molecular contents. This study aimed to explore the effects of human umbilical cord-derived mesenchymal stem cell exosomes (hucMSC-Exo) on subretinal fibrosis in vivo and in vitro and to investigate the anti-fibrotic mechanism of action of hucMSC-Exo.

Methods: In this study, human umbilical cord-derived mesenchymal stem cells (hucMSCs) were successfully cultured and identified, and exosomes were isolated from the supernatant by ultracentrifugation. A laser-induced choroidal neovascularization (CNV) and subretinal fibrosis model indicated that the intravitreal administration of hucMSC-Exo effectively alleviated subretinal fibrosis in vivo. Furthermore, hucMSC-Exo could efficaciously suppress the migration of retinal pigmented epithelial (RPE) cells and promote the mesenchymal-epithelial transition by delivering miR-27b-3p. The latent binding of miR-27b-3p to homeobox protein Hox-C6 (HOXC6) was analyzed by bioinformatics prediction and luciferase reporter assays.

Results: This study showed that the intravitreal injection of hucMSC-Exo effectively ameliorated laser-induced CNV and subretinal fibrosis via the suppression of epithelial-mesenchymal transition (EMT) process. In addition, hucMSC-Exo containing miR-27b repressed the EMT process in RPE cells induced by transforming growth factor-beta2 (TGF-β2) via inhibiting HOXC6 expression.

Conclusions: The present study showed that HucMSC-derived exosomal miR-27b could reverse the process of EMT induced by TGF-β2 via inhibiting HOXC6, indicating that the exosomal miR-27b/HOXC6 axis might play a vital role in ameliorating subretinal fibrosis. The present study proposed a promising therapeutic agent for treating ocular fibrotic diseases and provided insights into the mechanism of action of hucMSC-Exo on subretinal fibrosis.

Keywords: Epithelial–mesenchymal transition; Exosomes; Mesenchymal stem cells; Subretinal fibrosis.

Fig. 1

Characteristics of hucMSCs. a, b Cell morphology of passage 0 (bar = 50 μm) and passage 3 (bar = 200 μm) human umbilical cord mesenchymal stem cells (hucMSCs) observed under an inverted microscope (bar = 50 μm). c, d Osteogenic differentiation of hucMSCs was performed in the differentiation medium, while the control cells were grown in a regular medium. Both of them were dyed with Alizarin Red S staining. Most hucMSCs were Alizarin red positive. e, f Oil red O staining was used to stain hucMSCs after adipogenic differentiation, showing the Oil red O-positive lipid droplets. g Flow cytometry analysis of the phenotypic markers of hucMSCs showed that hucMSCs were positive for CD90, CD44, CD105, CD73, and CD105 and negative for CD34, CD45, CD11b, CD19, and human leukocyte antigen (HLA)-DR

Fig. 2

Identification of hucMSC-derived exosomes (hucMSC-Exo). a The hucMSC-Exo were isolated from the conditioned medium using serial centrifugation. b Transmission electron microscopic images of typical hucMSC-Exo. Scale bar = 100 nm. c Size distribution of the hucMSC-Exo was determined using a nanoparticle tracking analysis. hucMSC-Exo had an original concentration of 8.8e+9 particles/mL, a mean size of 159.0 nm, and a peak size of 128.6 nm. d Positive expression of markers CD9, TSG101, CD63, and HSP70 was mainly detected in hucMSC-Exo using Western blot analysis. e Fluorescence result of ARPE19 cells after being co-cultured with hucMSC-Exo labeled with PKH67, observed under a confocal microscope (scale bar = 25 μm)

Fig. 3

hucMSC-Exo treatment had less fibrosis development in vivo. a Study design. b The RPE–Bruch membrane complex was disrupted, the normal arrangement of the inner nuclear layer was lost and neovascular hide beneath the subretinal space 7 days after the laser burn. Compared with the PBS group, the number of collagen fibers reduced (blue) in the group treated with hucMSC-derived exosomes 35 days after laser photocoagulation. c Choroidal neovascularization (CNV; dyed with isolectin B4) and fibrosis (dyed with collagen type I) were recorded 7, 21, and 35 days after laser burn in the PBS and hucMSC-Exo groups. d Mean volume of the CNV and fibrosis 7, 21, and 35 days after laser burn

Fig. 4

Effects of hucMSC-Exo on cell migration. a, b Effect of different concentrations of hucMSC-Exo on the expression of occludin, Vimentin, N-cadherin, and α-SMA in TGF-β2-treated ARPE19 cells. c Typical morphological alteration of ARPE19 cells in the untreated group or in the 10 ng/mL TGF-β2-treated group. d Immunocytochemistry of ARPE19 cells. The cells were fixed with PFA and dyed with primary antibodies against ZO-1, α-SMA, and Vimentin. Scale bar = 20 μm. e Images of cells were recorded 0, 24, and 48 h after a scratch. Scale bar = 50 μm. f Harvested cells were plated on the upper chamber and migrated for an additional 12 h. The migrated cells were recorded under a phase-contrast microscope. Statistics are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Inhibitory effect of hucMSC-Exo on TGF-β2-treated EMT in ARPE19 cells. a Immunofluorescence staining of Vimentin, ZO-1, and a-SMA expression in ARPE19 cells treated with or without 100 μg/mL hucMSC-Exo; the nuclei were stained with DAPI. b Extracted total protein was loaded and analyzed by Western blot analysis. The relative protein expression was quantified. The data represent the average of three independent experiments. Statistics are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Relative miR-27b-3p expression in exosomes. a Candidate miRNA were screened using the Venn diagram package. b Predictions of eight potential targeted miRNAs of hucMSC-Exo were detected by qRT-PCR; HSF-Exo was used as the reference. Different-color areas represent different datasets. c Average miR-27b levels in hucMSC-Exo after treatments with RNase and Triton X-100 for 30 min, relative to miR-27b levels in the control untreated group, normalized to U6. Statistics are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

hucMSC-Exo were enriched with miR-27b-3p that could suppress the EMT process. a qRT-PCR showed that the application of antagomiR-27b-3p could counteract the overexpression of miR-27b-3p caused by hucMSC-Exo. b Western blot analysis indicated that antagomiR-27b-3p could recover the EMT process suppressed by hucMSC-Exo. c Effect of antagomiR-27b-3p on the EMT-related protein changes assessed by immunofluorescence. d–e Effects of antagomiR-27b-3p on the migration and wound-healing ability of ARPE19 cells were measured by Transwell and scratch assays; hucMSC-Exo was used as a reference. Statistics are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8

miR-27b-3p inhibited cell migration and expression of EMT-associated proteins in vitro. a miR-27b-3p level in three groups analyzed by qRT-PCR. b Effect of miR-27b-3p on the expression of EMT-related proteins occludin, Vimentin, N-cadherin, and α-SMA evaluated by Western blot analysis. c Immunofluorescence was further used to assess cell morphological alteration. d–e Effects of miR-27b-3p on the migration and wound-healing ability of ARPE19 cells. Statistics are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 9

miR-27b-3p modulated the EMT process by suppressing HOXC6. a Potential targets of miR-27b-3p were predicted using five databases (TargetScan, miRanda, miRtarbase, miRSystem, and miRDB). b The targeted relationship of miR-27b-3p with HOXC6 was verified by the dualluciferase assay. c Effect of miR-27b-3p on the expression of HOXC6 assessed by Western blot analysis. Data are the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 10

Schematic representation portrays that hucMSC-derived exosomal miR-27b-3p could alleviate subretinal fibrosis via suppressing epithelial–mesenchymal transition by targeting HOXC6