Regulation of exosomes derived from miR-34a overexpression of adipose derived stem cells effects on proliferation and apoptosis of human hypertrophic scar fibroblasts

Acta Universitatis Medicinalis Anhui 2020 06 v.55 887-893     font:big middle small

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Authors:Xiao Xiangyang; Zheng Deyi; Wang Baoyun

Keywords:adipose derived stem cells;exosome;miR-34a;human hypertrophic scar fibroblasts;proliferation and apoptosis

DOI:10.19405/j.cnki.issn1000-1492.2020.06.013

〔Abstract〕 Objective To investigate the effect of exosomes(Exo) released from adipose derived stem cells(ADSCs) with overexpressed miR-34 a on proliferation and apoptosis of human hypertrophic scar fibroblasts(HSF). Methods Primary ADSCs were isolated from human adipose tissue and their surface markers were identified by flow cytometry. The osteogenic differentiation of ADSCs was observed by using alizarin red and alkaline phosphatase staining. ADSCs were infected by lentiviruses containing miR-34 a to obtain over-expressed miR-34 a ADSCs(ADSCs/miR-34 a) and negative control ADSCs(ADSCs/miR-NC). The exosome was extracted from cell supernatant of each group and ADSCs exosome(ExoADSCs), over-expressed miR-34 a ADSCs exosome(ExoADSCs/miR-34 a) and negative control ADSCs exosome(ExoADSCs/miR-NC) were obtained. The morphology of exosomes was observed by transmission electron microscope(TEM), and the surface marker proteins CD81 and CD63 were detected by Western blot. The exosomes were co-cultured with HSF and divided into control group(HSF+PBS), Exo ADSCs treatment group(HSF+ExoADSCs), ExoADSCs/miR-NCtreatment group(HSF+ ExoADSCs/miR-NC) and ExoADSCs/miR-34 atreatment group(HSF+ExoADSCs/miR-34 a). Quantificational real-time PCR(qRT-PCR) was used to detect the expression of miR-34 a in ADSCs and exosomes. The proliferation of HSF cells was detected by MTT assay. The apoptotic rate of HSF cells was detected by flow cytometry. The expression levels of apoptosis related proteins(cleaved-Caspase-3, Bax and Bcl-2), fibrosis-related proteins(COL Ⅰ, COL Ⅲ) and TGF-β/Smad signal pathway related proteins(TGF-β1, p-Smad 2 and p-Smad3) were detected by Western blot assay. Results ADSCs were successfully isolated and possessed osteogenic differentiation ability. TEM showed round or oval vesicle bodies with a diameter of 50~100 nm with bilayer membrane structure. Western blot showed positive expression of CD81 and CD63, which were the marker proteins of exosomes. Overexpression of miR-34 a could increase the expression of miR-34 a in ADSCs and exosomes. Compared with ExoADSCsgroup and ExoADSCs/miR-NCgroup, ExoADSCs/miR-34 aintervention could inhibit HSF cell proliferation(P<0.05), increase cell apoptosis rate(P<0.05), up-regulate the expression of cleaved-Caspase-3 and Bax protein(P<0.05), and down-regulate the expression of Bcl-2, COL Ⅰ, COL Ⅲ, p-Smad2, p-Smad3 and TGF-β1 protein(P<0.05). Conclusion The exosome of miR-34 a overexpressed ADSCs may inhibit the HSF cell proliferation and promote cell apoptosis by down-regulating TGF-β/Smad signaling pathway.