The Effect And Mechanism Of Amniotic Mesenchymal Stem Cell-derived Exosomes Carried OIP5-AS1 Targeting MiR-29a-3p To Regulate SIRT1 In Promoting Diabetic Wound Healing By Angiogenesis

Background:The diabetic wound is that the skin of diabetic patients were damaged because of skin ulcers and trauma,which is one of the common complications in diabetic patients and easy to result in paresthesia and functional change of the corresponding parts.There are about 20% of diabetic patients accompanied with diabetic wounds so far and the number is still increasing year by year.The current therapeutic methods,such as debridement,dressing change,bioengineering skin,negative pressure wound treatment and flap repair,are difficult to obtain the ideal results.The unhealed wound leads to the development of the diabetic wound,which brings many difficulties for clinical therapy.Previous research demonstrated that the angiogenesis of diabetic wounds was impaired by hyperglycemia,thus delaying the healing process.It has important clinical significance to promote the angiogenesis of diabetic wound.Many studies have suggested that exosomes can carry active molecules to accelerate diabetic wound healing by angiogenesis.However,human amniotic mesenchymal stem cell exosomes(hAMSC-Exos)can promote the diabetic wound healing by angiogenesis remains unclear and the underlying mechanisms of angiogenesis facilitated by hAMSC-exos in the healing of diabetic wounds needs to be further explored.Objective:In this study,we sought to investigate The effect and mechanism of amniotic mesenchymal stem cell-derived exosomes carried OIP5-AS1 targeting miR-29a-3p to regulate SIRT1 in promoting diabetic wound healing by angiogenesis,so as to provide new ideas and theoretical basis for the treatment of diabetic wounds.Methods:1.The human amniotic tissues were separated from maternal placentas and the hAMSCs were isolated by trypsin-EDTA combined with collagenase II.The biomarkers of hAMSCs were identified by flow cytometry and the multipotential differentiation of hAMSCs were identified by osteogenic and adipogenic differentiation.The culture supernatant of hAMSCs was collected to extract hAMSC-Exos by ultracentrifugation and the hAMSC-Exos were identified by transmission electron microscope(TEM)and western blot.The discarded umbilical cord tissue was digested by trypsin-EDTA combined with collagenase II to isolate the primary HUVECs,then detected the expression of CD31 by fluorescence confocal microscopy.The uptake of hAMSC-Exos labeled with PKH26 by HUVECs were observed.The effects of hAMSC-Exos on proliferation,migration and tube formation of HUVECs treated with high glucose were determined by CCK-8,scratch,Transwell and tube-formation assay.Diabetic wound model was established to observe the effect of hAMSC-Exos on diabetic wound healing.2.Firstly,the differentially expressed lncRNAs and mRNAs between hAMSCs and hAMSC-Exos were detected by Arraystar Human Lnc RNA Microarray.Subsequently,GO and KEGG pathway analyses were performed for differentially expressed mRNAs,and the reliability of the microarray results was verified by RT?q PCR.Finally,the differentially expressed lncRNAs associated with angiogenesis in hAMSC-Exos were selected to construct the ceRNA networks.3.The subcellular localization of OIP5-AS1 in HUVECs and the expression of OIP5-AS1,miR-29a-3p and SIRT1 in diabetic wound tissue and HUVECs treated with high glucose were detected by RT-PCR.4.The transfection efficiency of oe-OIP5-AS1,si-OIP5-AS1,miR-29a-3p mimics,miR-29a-3p inhibitor,oe-SIRT1 and si-SIRT1 were detected by RT-PCR after they were transfected into HUVECs treated with high glucose.The mRNA expression levels of downstream target genes miR-29a-3p and SIRT1 were detected by RT-PCR and the protein expression levels of SIRT1 were detected by western blot.5.The effects of oe-OIP5-AS1,si-OIP5-AS1,miR-29a-3p mimics,oe-SIRT1 and si-SIRT1 transfection on proliferation,migration and tube formation of HUVECs treated with high glucose were determined by CCK-8,scratch,Transwell and tube-formation assay.6.The target relationship between OIP5-AS1 and miR-29a-3p,miR-29a-3p and SIRT1 were verified by the dual luciferase reporter gene assay after the binding site between OIP5-AS1 and miR-29a-3p,miR-29a-3p and SIRT1 was predicted by RNAhybrid and Target Scan websites.7.si-OIP5-AS1 and oe-SIRT1 were co-transfected into HUVECs,and cell recovery experiments were conducted to determine whether the oe-SIRT1 could save the effect of down-regulated OIP5-AS1 on the inhibition of proliferation,migration and tube formation of HUVECs.8.The phosphorylation levels of PI3K/AKT signaling pathway related proteins in HUVECs treated with high glucose or transfected with oe-OIP5-AS1 was detected by western blot.Results:1.The cells isolated from amniotic tissues strongly expressed the MSC marker proteins,CD44,CD73,CD90,and CD105,but did not express the hematopoietic stem cell marker proteins,CD34,and CD45,or the histocompatibility protein,HLA-DR and they could be induced to differentiate into osteoblasts and adipocytes,respectively,in vitro,which demonstrated the isolated cells were hAMSCs.The Flow Nano Analyzer revealed that the diameters of hAMSC-Exos were within the range of30-150 nm.Western blot analysis demonstrated that the hAMSC-Exos expressed the exosomal surface markers,CD9,CD63 and CD81.2.The cells isolated from umbilical vein tissue strongly expressed CD31,which demonstrated the isolated cells were HUVECs.The hAMSC-Exos labeled with PKH26 could be transferred to the HUVECs.The hAMSC-Exos could facilitate the proliferation,migration and the angiogenic activities of HUVECs treated with high glucose levels by CCK-8,scratch,transwell and tube-formation assay in vitro.And they could facilitate diabetic wound healing by angiogenesis in vivo.3.The differentially expressed lncRNAs and mRNAs in hAMSC-Exos were screened by microarray.The results of GO and KEGG Pathway analysis suggest that hAMSC-Exos may regulate the construction of cell membrane,recognition of cell molecules and receptors and transfer of information between cells,which may play dominant roles in wound repair and tissue regeneration.The RT?q PCR results means that the data acquired by the microarray were reliable.The lncRNAs PANTR1,H19,OIP5-AS1 and NR2F1-AS1 associated with angiogenesis were selected to construct the ceRNA networks,which may participate in regulating the healing process of diabetic wounds.4.The results of RT-PCR suggested that the expression of OIP5-AS1 and SIRT1 decreased in diabetic wound tissue and HUVECs treated with high glucose,while the expression of miR-29a-3p increased.The experiment of nucleoplasmic separation confirmed that OIP5-AS1 mainly distributed in the cytoplasm of HUVECs.5.Oe-OIP5-AS1 promoted proliferation,migration and tube formation of HUVECs treated with high glucose by CCK-8,scratch,transwell and tube-formation assay in vitro.Si-OIP5-AS1 inhibited proliferation,migration and tube formation of HUVECs.Oe-OIP5-AS1 inhibited the expression of miR-29a-3p and promoted the expression of SIRT1 mRNA and protein,and si-OIP5-AS1 promote the expression of miR-29a-3p and inhibited the expression of SIRT1 mRNA and protein.6.miR-29a-3p mimics inhibited proliferation,migration and tube formation of HUVECs treated with high glucose by CCK-8,scratch,transwell and tube-formation assay in vitro.But miR-29a-3p inhibitor promoted proliferation,migration and tube formation of HUVECs.miR-29a-3p mimics inhibited the expression of SIRT1 mRNA and protein but miR-29a-3p inhibitor promoted the expression of SIRT1 mRNA and protein.7.Oe-SIRT1 promoted proliferation,migration and tube formation of HUVECs treated with high glucose by CCK-8,scratch,transwell and tube-formation assay in vitro.Si-SIRT1 inhibited proliferation,migration and tube formation of HUVECs.Oe-SIRT1 promoted the expression of SIRT1 mRNA and protein,and si-SIRT1 inhibited the expression of SIRT1 mRNA and protein.8.The binding site between OIP5-AS1 and miR-29a-3p or SIRT1 and miR-29a-3p was verified by dual luciferase reporter gene assay,which showed that the luciferase activity of OIP5-AS1 or SIRT1 WT was inhibited by miR-29a-3p mimics.However miR-29a-3p mimics had no effect on the luciferase activity of OIP5-AS1 MUT and SIRT1 MUT.It is suggested that OIP5-AS1 sponged miR-29a-3p、miR-29a-3p sponged SIRT1 to regulate the expression of miR-29a-3p and SIRT1.9.The results of cell recovery experiments showed that the proliferation,migration and tube formation of HUVECs in si-OIP5-AS1+oe-SIRT1 group were significantly enhanced compared with that in si-OIP5-AS1+oe-NC group,which suggested that oe-SIRT1 can save the effect of si-OIP5-AS1 on the proliferation,migration and tube formation of HUVECs.10.The phosphorylation levels of PI3K/AKT signaling pathway related proteins in HUVECs treated with high glucose was significantly increased after transfection of oe-OIP5-AS1,but there were no differences after transfection of oe-NC.These results indicated that OIP5-AS1 ameliorated the injury of HUVECs induced by high glucose through the activation of PI3K/AKT signaling pathway.Conclusion:1.Our results demonstrated that hAMSC-Exos can facilitate the proliferation,migration and angiogenic activities of HUVECs treated with high glucose and facilitate diabetic wound healing by angiogenesis.2.The effect and mechanism of amniotic mesenchymal stem cell-derived exosomes carried OIP5-AS1 targeting miR-29a-3p to regulate the expression of SIRT1 and activate PI3K/AKT signaling pathway to meliorate the injury of HUVECs induced by high glucose,thus to promotes proliferation,migration,and tube formation of HUVECs treated with high glucose.