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. 2021 Aug 2;10(10):e12124. doi: 10.1002/jev2.12124

FIGURE 4.

FIGURE 4

miRNA cargo in HBEC EVs attenuates TGF‐β‐induced myofibroblast differentiation via suppression of WNT5A and WNT10B expression. a Ingenuity Pathway Analysis (IPA) of upstream regulators of WNT ligand genes in LFs treated for 48 h with HBEC EVs. b qRT‐PCR analysis of WNT1, WNT3A, WNT5A and WNT10B mRNAs in LFs treated for 24 h with HBEC EVs (10 μg/ml, n = 3). *P < 0.05. c Representative immunoblot and quantitative analysis showing the amount of type I collagen, α‐ SMA, and β‐actin in LFs treated for 24 h with PBS or HBEC EVs (10 μg/ml) in the presence or absence of WNT5A (200 pg/ml, lane 2,3) and WNT10B (200 pg/ml, lane 4,5). Protein samples were collected after 72 h of stimulation. *P < 0.05, **P < 0.005. d, e WNT5A and WNT10B evaluation value in the mRNA microarray dataset GSE53845 from the Gene Expression Omnibus (GEO) database. *P < 0.05, **P < 0.005. f Venn diagram of the candidate miRNAs targeting WNT5A and WNT10B from TargetScan database. g, h qRT‐PCR analysis of WNT5A and WNT10B mRNAs in LFs transfected with candidate target miRNA mimics or negative control. *P < 0.05, **P < 0.005. i Representative immunoblot analysis showing the amount of type I collagen, α‐ SMA, and β‐actin in LFs treated with validated miRNA mimics or negative control in the presence or absence of TGF‐β1 (2 ng/ml). j qRT‐PCR analysis of cellular miRNA expression in LFs incubated for 24 h with HBEC EVs (10 μg/ml, n = 3) or PBS. *P < 0.05, **P < 0.005, ***P < 0.001