Circulating Exosomal MiR-20b-5p Inhibition Restores Wnt9b Signaling And Reverses Diabetes-Associated Impaired Wound Healing

Part ?.Diabete-Exos and Normal-Exos isolation and functional verification.Objective: As a kind of new nanoscale biomaterial and drug carrier,exosome has attracted accumulative attention for its regulatory effect on biological behavior in vivo.The prolonged wound healing of diabetic wounds carried in great burdens for the patients,which has been a great challenge in clinic.It was reported that a huge volume of exosomes exist in the circulation.We aimed to identify a new therapeutic target for diabetic wound by investigating the biological behavior of exosomes in patients' circulation.Method: The ultra-centrifugal method was used to isolate the exosomes in different groups.Then,TEM was taken to catch the morphology of exosomes.DLS was used to assess the size of exosomes.And WB was used to identify the bio-markers CD 81 and TSG 101 of exosomes.Furthermore,The fluorescent dye PKH26 was used to perform the exosome intake test.Moreover,the effects of exosomes on the proliferation ability of HUVECs were measured using EDU fluorescence staining,CCK-8,WB,qRT-PCR,and flow cytometry assays.The effects of exosomes on HUVECs apoptosis were observed by flow cytometry,WB,and qRT-PCR analyses.The effect of exosomes on HUVECs tube formation ability was assessed using cell tube formation assay.In addition,the effect of exosomes on wound healing was assessed by producing a murine wound model.The blood perfusion of the wound was observed using small animal Doppler examination,and the neovascularization of the wound was assessed by the CD31 immunohistochemical test of the skin at the wound.Results: The morphology of exosomes in the two groups was disk-shaped or hemispherical,and DLS showed that the particle sizes of exosomes in the two groups were ranging from30 to 150 nm.Both exosomes in the two groups were identified by WB analysis with double positive markers CD 81 and TSG 101.PKH26 intake assay confirmed that exosomes in both groups could be taken up by HUVECs.Diabete-Exos could significantly inhibit HUVECs survival and tube formation with a concentration-dependent manner.Animal experiments indicated that Diabete-Exos could significantly delay the healing of the wound in the back of mice,reduce the amount of blood perfusion and inhibit the new vessels formation at the wound,and this regulation showed changes in concentration dependent.Conclusion: Diabete-Exos can be taken up by HUVECs,and exerts a harmful effect on the survival and functionality of HUVECs,leading to the decreased neovascularization and blood perfusion at the wound,and thus delaying wound healing.Part ?.The effect of exosomal miR-20b-5p on wound healingObjective: miR-20b-5p has previously been demonstrated to be enriched in Diabete-Exos,but the effect of this exosome-derived miR-20b-5p P on diabetic wounds healing has not been reported previously.We sought to explore the function of this exosome-derived miR-20b-5p to discover the potential mechanism of the effect of Diabete-Exos on wound healing.Methods: MiR-20b-5p expression in Normal-Exos and Diabete-Exos was assessed by qRT-PCR analysis,and the expression of miR-20b-5p in HUVECs with Diabete-Exos or Normal-Exos treated was also evaluated.The effect of Diabete-Exos with miR-20b-5p knock down on the survival of HUVECs was detected by CCK-8,EDU fluorescence staining,WB,qRT-PCR,and flow cell cycle assays,respectively.Similarly,the effect of Diabete-Exos with miR-20b-5p knock down on the tube formation ability of HUVECs was detected by cell tube formation test.The effects of miR-20b-5p on HUVECs function,apoptosis,and tube formation ability were measured by CCK-8,EDU fluorescence staining,WB,qRT-PCR,flow cytometry and tube formation assays.Diabetic murine model was produced based on the STZ injection method,and the miR-20b-5p knockout diabetes mice were obtained by the hybrid method.The effect of miR-20b-5p on the wound healing was measured by the general wound closure view,small doppler examination,and the CD31 immunohistochemical detection.Results: qRT-PCR results showed that miR-20b-5p expression in HUVECs with Diabete-Exos treatment was markedly higher than other groups,and the harmful effect of Diabete-Exos on HUVECs was significantly alleviated after the deletion of miR-20b-5p in exosomes.Inhibition of miR-20b-5p was capable to significantly enhance the proliferation and angiogenesis of HUVECs,and delay its apoptosis rate.In animal experiments,the results showed that the inhibition of miR-20b-5p could markedly accelerate the wound healing,elevate the blood volume perfusion of the wound,and increase the new vessels formation at the wound.The wound healing rate,blood flow perfusion and new blood vessels of diabetic mice after the deletion of miR-20b-5p were markedly better than normal diabetic mice.Conclusion: Inhibition of the miR-20b-5p expression in Diabete-Exos could significantly alleviate the adverse effects of Diabete-Exos on HUVECs and wound healing,and the abnormally high level of miR-20b-5p in Diabete-Exos is the potential mechanism of its adverse effect on wound healing.Part ?.The underlying mechanism of exosomal miR-20b-5p on regulation of wound healing.Objective: Exosomal miR-20b-5p has been demonstrated to exist a robust regulatory effect on HUVECs and diabetic wounds,and the potential mechanism of this regulation needs to be investigated.Methods: The potential downstream mechanism of miR-20b-5p was predicted using the online predicting tools,including miRBase,Target Scan and miRanda.The binds between miR-20b-5p and Wnt9 b was then verified using dual luciferase assay.Furthermore,the qRT-PCR analysis was used to assess the level of Wnt9 b in HUVECs with agomiR-20b-5p or antagomiR-20b-5p treatment.Moreover,EDU fluorescence staining,WB,flow cytometry,and tube formation assay were used to explore the effect of si RNA Wnt9 b on the survival and functionality of HUVECs.Results: Wnt9 b is one of the potential downstream mechanism of miR-20b-5p,and then,the dual luciferase assay confirmed that miR-20b-5p was able to bind closely with Wnt9 b.Furthermore,the qRT-PCT analysis indicated that overexpression of miR-20b-5p can significantly inhibit the Wnt9 b expression.WB showed that silencing Wnt9 b can significantly inhibit Wnt9 b expression and ?-catenin expression,as well as reducing the survival and tube formation ability of HUVECs.Conclusion: Wnt9b/?-catenin signaling is an important potential molecular mechanism of exosomal miR-20b-5p in the regulation of wound healing.