Microvesicles derived from human Wharton's jelly mesenchymal stem cells enhance autophagy and ameliorate acute lung injury via delivery of miR-100

Abstract

Objectives: Microvesicles (MVs) derived from human Wharton's jelly mesenchymal stem cells (MSC-MVs) were demonstrated to ameliorate acute lung injury (ALI). We have previously found that MSC-MV-transferred hepatocyte growth factor was partly involved in their therapeutic effects. Since MSC-MVs also contained a substantial quantity of miR-100, which plays an important role in lung cancer and injury, we speculated that miR-100 might similarly account for a part of the therapeutic effects of MSC-MVs.

Methods: MSCs were transfected with miR-100 inhibitor to downregulate miR-100 in MSC-MVs. A rat model of ALI and cell injury in rat type II alveolar epithelial cell line (L2) was induced by bleomycin (BLM). A co-culture model of alveolar epithelial cells and MSC-MVs was utilized to examine the therapeutic role of MSC-MVs and mechanism.

Results: MSC-MV treatment attenuated BLM-induced apoptosis and inflammation in BLM-treated L2 cells and ameliorated BLM-induced lung apoptosis, inflammation, and fibrosis in BLM-induced ALI rats. The beneficial effect of MSC-MVs was partly eliminated when miR-100 was knocked down in MSCs. Moreover, MSC-MV-transferred miR-100 mediated the therapeutic effect of MSC-MVs in ALI through enhancing autophagy by targeting mTOR.

Conclusion: MSC-MVs enhance autophagy and ameliorate ALI partially via delivery of miR-100.

Keywords: Acute lung injury; Autophagy; Mesenchymal stem cells; Microvesicles; miR-100.

Fig. 1

MSC-MVs mediate transfer of miR-100 to BLM-treated L2 cells. a qRT-PCR analysis of miR-100 expression in MSCs and MSC-MVs. b qRT-PCR analysis of miR-100 expression in NCI-transfected MSCs and miR-100I-transfected MSCs. c qRT-PCR analysis of miR-100 expression in MVs derived from NCI-transfected MSCs (NCI-MVs) and MVs from miR-100I-transfected MSCs (miR-100I-MVs). d The MVs derived from FAM (green)-miR-100-transfected MSCs were labeled with Dil (red) and co-cultured with rat type II alveolar epithelial cell line L2 (nuclei stained with Hoechst 33342, blue). The distribution and intensity of fluorescence were observed by confocal laser microscopy to analyze MV uptake by L2 cells. e qRT-PCR analysis of miR-100 expression in L2 cells in the groups of control, BLM, BLM+MVs, BLM+NCI-MVs, and BLM+miR-100I-MVs. Data are presented as the means ± SD (n = 3). **p < 0.01, vs. MSC; ^##p < 0.01, vs. NCI; ^$$p < 0.01, vs. NCI-MVs; ^&&p < 0.01, vs. control; ^@@p < 0.01, vs. BLM; ^%p < 0.05, vs. BLM+NCI-MVs

Fig. 2

MSC-MVs attenuate the BLM-induced apoptosis and inflammation via transferring miR-100. a, b Annexin V-FITC/PI apoptotic assay (a) and levels of IL-6, IL-8, and TNF-α in cell supernatant (b) in L2 cells in the groups of control, BLM, BLM+mimic NC, and BLM+miR-100 mimic. c, d Annexin V-FITC/PI apoptotic assay (c) and levels of IL-6, IL-8, and TNF-α in cell supernatant (d) in L2 cells in the groups of control, BLM, BLM+MVs, BLM+NCI-MVs, and BLM+miR-100I-MVs. Data are presented as the means ± SD (n = 3). **p < 0.01, vs. control; ^##p < 0.01, vs. BLM+mimic NC; ^$$p < 0.01, vs. BLM; ^&&p < 0.01, vs. BLM+NCI-MVs

Fig. 3

MSC-MVs restore the BLM-inhibited autophagy via transferring miR-100 targeting mTOR. a The putative miR-100 binding sites in mTOR (mTOR WT) or and the designed mutant sequence (mTOR Mut) were indicated. Luciferase reporter assay was conducted to evaluate the interaction between mTOR 3′-UTR and miR-100. b The protein levels of mTOR and autophagy-related LC3-I, LC3-II, and Beclin-1 in L2 cells in the groups of control, BLM, BLM+MVs, BLM+NCI-MVs, and BLM+miR-100I-MVs. Data are presented as the means ± SD (n = 3). **p < 0.01, vs. mimic NC; ^##p < 0.01, vs. control; ^$p < 0.05, ^$$p < 0.01, vs. BLM; ^&p < 0.05, ^&&p < 0.01, vs. BLM+NCI-MVs

Fig. 4

MSC-MVs attenuate the BLM-induced apoptosis and inflammation via elevating autophagy. The protein levels of autophagy-related LC3-I, LC3-II, and Beclin-1 (a); levels of IL-6, IL-8, and TNF-α in cell supernatant (b); and annexin V-FITC/PI apoptotic assay (c) in L2 cells in the groups of BLM, BLM+MVs, BLM+MVs+DMSO, and BLM+MVs+MHY1485. Data are presented as the means ± SD (n = 3). **p < 0.01, vs. BLM; ^##p < 0.01, vs. BLM+MVs+DMSO

Fig. 5

Changes of miR-100 expression, autophagy level, lung apoptosis, and inflammation in ALI rats 48 h following administration with miR-100I-MVs. qRT-PCR analysis of miR-100 in lung tissues (a); western blot analysis of protein levels of mTOR, LC3-I, LC3-II, and Beclin-1 in lung tissues (b); total protein level in BALF (c); total cell numbers in BALF (d); neutrophil counts in BALF (e); levels of IL-6, IL-8, and TNF-α in BALF determined by ELISA (f); and TUNEL staining of apoptotic lung tissue cells (g) from ALI rats following 48 h treatment of vehicle (PBS), MVs, NCI-MVs, and miR-100I-MVs. N = 10 in each group. **p < 0.01, vs. control; ^##p < 0.01, vs. ALI+vehicle; ^$p < 0.05, ^$$p < 0.01, vs. ALI+NCI-MVs

Fig. 6

Changes of miR-100 expression, autophagy level, lung fibrosis, and inflammation in ALI rats 1 week following administration with miR-100I-MVs. qRT-PCR analysis of miR-100 in lung tissues (a); western blot analysis of protein levels of mTOR, LC3-I, LC3-II, and Beclin-1 in lung tissues (b); total protein level in BALF (c); total cell numbers in BALF (d); neutrophil counts in BALF (e); levels of IL-6, IL-8, and TNF-α in BALF determined by ELISA (f), and lung fibrosis indicated by Masson’s trichrome staining of lung tissues (g) from ALI rats following 1 week treatment of vehicle (PBS), MVs, NCI-MVs, and miR-100I-MVs. N = 10 in each group. **p < 0.01, vs. control; ^##p < 0.01, vs. ALI+vehicle; ^$p < 0.05, ^$$p < 0.01, vs. ALI+NCI-MVs

Fig. 7

MSC-MVs ameliorate BLM-induced lung injury via transferring miR-100 in vivo. Representative histopathological changes of lung injury in ALI rats 1 week following administration with miR-100I-MVs. The upper and lower panels are magnification, × 200 and × 400, respectively. The injury was scored with a semi-quantitative grading system. **p < 0.01, vs. control; ^#p < 0.01, vs. ALI+vehicle; ^$$p < 0.01, vs. ALI+NCI-MVs