Micro-vesicles from mesenchymal stem cells over-expressing miR-34a inhibit transforming growth factor-β1-induced epithelial-mesenchymal transition in renal tubular epithelial cells in vitro

Background:The use of microRNAs in the therapy of kidney disease is hampered by the difficulties in their effective delivery.Microvesicles(MVs)are known as natural carriers of small RNAs.Our prior research has demonstrated that MVs isolated from mesenchymal stem cells(MSCs)are capable of attenuating kidney injuries induced by unilateral ureteral obstruction and 5/6 sub-total nephrectomy in mice.The present study aimed to evaluate the effects of miR-34a-5p(miR-34a)-modified MSC-MVs on transforming growth factor(TGF)-β1-induced fibrosis and apoptosis in vitro.Methods:Bone marrow MSCs were modified by lentiviruses over-expressing miR-34a,from which MVs were collected for the treatment of human Kidney-2(HK-2)renal tubular cells exposed to TGF-β1(6 ng/mL).The survival of HK-2 cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)and Annexin V-Light 650/propidium iodide(PI)assays.The expression levels of epithelial markers(tight junction protein 1[TJP1]and E-cadherin)and mesenchymal markers(smooth muscle actin alpha(α-SMA)and fibronectin)in HK-2 cells were measured using Western blot analysis and an immunofluorescence assay.In addition,changes in Notch-1/Jagged-1 signaling were analyzed using Western blotting.Data were analyzed using a Student’s t test or one-way analysis of variance.Results:MiR-34a expression increased three-fold in MVs generated by miR-34a-modified MSCs compared with that expressed in control MVs(P<0.01,t=16.55).In HK-2 cells,TJP1 and E-cadherin levels decreased to 31%and 37%after treatment with TGF-β1,respectively,and were restored to 62%and 70%by miR-34a-enriched MSC-MVs,respectively.The expression ofα-SMA and fibronectin increased by 3.9-and 5.0-fold following TGF-β1 treatment,and decreased to 2.0-and 1.7-fold after treatment of HK-2 cells with miR-34a-enriched MSC-MVs.The effects of miR-34a-enriched MSC-MVs on epithelial-mesenchymal transition(EMT)markers were stronger than control MSC-MVs.The effects of miR-34a-enriched MSC-MVs on these EMT markers were stronger than control MSC-MVs.Notch-1 receptor and Jagged-1 ligand,two major molecules of Notch signaling pathway,are predicted targets of miR-34a.It was further observed that elevation of Notch-1 and Jagged-1 induced by TGF-β1 was inhibited by miR-34a-enriched MSC-MVs.In addition,TGF-β1 exposure also induced apoptosis in HK-2 cells.Although miR-34a-mofidied MSC-MVs were able to inhibit TGF-β1-triggered apoptosis in HK-2 cells,the effects were less significant than control MSC-MVs(control:TGF-β1:miR-nc-MV:miR-34a-MV=1.3:0.6:1.1:0.9 for MTT assay,1.8%:23.3%:9.4%:17.4%for apoptosis assay).This phenomenon may be the result of the pro-apoptotic effects of miR-34a.Conclusions:The present study demonstrated that miR-34a-over-expressing MSC-MVs inhibit EMT induced by pro-fibrotic TGF-β1 in renal tubular epithelial cells,possibly through inhibition of the Jagged-1/Notch-1 pathway.Genetic modification of MSC-MVs with an anti-fibrotic molecule may represent a novel strategy for the treatment of renal injuries.