Effects Of Skeletal Muscle Exosome On Endothelial Cell Proliferation And MicroRNA Sequencing Analysis

Background:As for diabetics,diabetic foot（DF）is one of the serious complications and there will happen an amputation every 20 seconds.Lower limb ischemia is a major element of high amputation rate of DF.Improving limb blood supply can be an important way to treat DF and reduce amputation rates.Skeletal muscle is the main component of the lower extremities,rich in mitochondria,which is very sensitive to the changes of ischemia and hypoxia.At the same time,skeletal muscle can participate in metabolic process of tissue and cell by secreting exosomes.Therefore,skeletal muscle can be used as a target organ to study the compensatory response of diabetic foot.Exosomes carry a variety of substances such as nucleic acids（including microRNA,lncRNA,circRNA）,cytokines and proteins,which mediate intercellular signal transduction and regulate pathophysiological reactions.Studies of exosome microRNAs have reported that the ischemia and oxygen deficiency conditions will be reversed by neovascularization,which is particularly important in ischemic cardiovascular and cerebrovascular diseases.However,there is no report on the role of exosome microRNAs in ischemic DF.In conclusion,we mainly devote ourselves to studying the function and mechanism of skeletal muscle cells in regulating angiogenesis under hypoxic conditions.This study will provide theoretical and experimental basis for the discovery of new DF therapeutic targets in the future.Objective:Through hypoxia culture in vitro to simulate lower limb ischemia.The vascular endothelial cells were treated with exosomes secreted by skeletal muscle cells cultured under hypoxia and non-hypoxia conditions respectively.Then the related processes of angiogenesis were observed,so as to clarify the role of skeletal muscle cell exosomes cultured under hypoxia in angiogenesis.Furthermore,the expression profiles of microRNA in exosomes of skeletal muscle cells were further analyzed under hypoxia condition,in order to reveal the molecular mechanism of the involvement of skeletal muscle cells in regulating angiogenesis.Methods:1.Skeletal muscle cells were cultured under hypoxia incubator.2.Exosomes were extracted by ultracentrifugation.TEM and western blotting were performed to identify exosomes.3.Though fluorescence microscopy to observe whether Dil labeled exosomes entered HUVEC cells.4.CCK-8,cell scratch and angiogenesis test were used to detect the effects of exosomes on the function of HUVEC cells.5.After generating microRNA sequencing library from HSMC exosomes under hypoxia（treated）and non-hypoxia（control）conditions,the highly sensitive Agilent Bioanalyzer 2100 system were used to detect library quality.Then microRNA cluster analysis was performed.Results:1.Identification of exosomes from skeletal muscle cell.Under TEM,we observed that exosomes were round or oval,with pale central color and clear in edges,with diameters ranging from 30 to 150nm.Western Blot showed that proteins Alix and CD63 were positive in both skeletal muscle cells and skeletal muscle cell derived exosomes.The results indicated that HSMC exosomes were successfully isolated after ultracentrifugation.2.Exosome tracing observation results.Through the fluorescence microscope,we observed that Dil labeled HSMC exosome were absorbed by HUVEC cells,suggesting that exosomes could enter HUVEC cells.3.Effects of exosomes on proliferation,migration and tube formation of endothelial cells.The results of CCK-8 experiment,scarification test and tube forming experiment suggested that anoxic HSMC exosomes can promote the proliferation,migration and tube formation of HUVEC cells（P<0.05）.4.Changes of microRNA expression profile in exosomes.11 differentially expressed microRNAs were found in exosomes.Compared with hypoxic HSMC exosomes,5 of them were up regulated,including hsa-miR-7641,hsa-miR-29a-5p,hsa-let-7i-3p,hsa-let-7f-1-3p and hsa-miR-3607-3p,6 of them were down regulated,including hsa-miR-615-3p,hsa-miR-128-3p,has-miR-210-3p and novel₁96,has-miR-125a-3p,hsa-miR-10a-3p.Conclusions and significations:1.HSMC derived exosomes can promote the proliferation,migration and tube formation of vascular endothelial cells under hypoxia.2.We also found changes of microRNA expression profile in HSMC exosomes of hypoxic compared to anoxic,5 microRNAs were up-regulated,including hsa-miR7641,hsa-miR-29a-5p,hsa-let-7i-3p,hsa-let-7f-1-3p and hsa-miR-3607-3p,6 microRNAs were down regulated,including hsa-miR-615-3p,hsa-miR-128-3p,hsamiR-210-3p and novel₁96,hsa-miR-125a-3p,hsa-miR-10a-3p.These results will provide theoretical basis and new direction to the prevention and treatment of ischemic DF.