Study Of Mechanism Of Browning Of White Adipose Tissue By Exercise-induced In Mice Through Exosome CircRNA₀011487-miR328/PRDM16 Axis

Objective:Exercise can promote the browning of white adipose tissue,improve the energy consumption of the body,accelerate the metabolism of glucose and fat and achieve the purpose of weight loss.Because of the physiological,biochemical and physiological changes caused by exercise state and long stage after exercise have significant effects on the body,little study about browning of white adipose tissue induced by different exercise periods have been reported,and there are few reports on the browning of white adipose tissue gene in mice induced by exercise.In this study,a new mechanism of exercise promoting browning of white adipose tissue was explored through exercise training in mice.Methods:1^stt PartBALB/C mice exercised for 4 weeks to analyze the effect of exercise on browning of white adipose tissue in mice.The plasma samples were collected after the last exercise for 24 hours,and the exosome were isolated by ultracentrifugation.The exosome were identified by transmission electron microscope and western immunoblotting.The effect of exosome on white adipocytes after exercise was evaluated by primary culture of white adipocytes in mice.The difference of circRNA between exosome of sedentary and exosome after exercise was analyzed by high throughput sequencing.The expression level of circRNA in exosome and tissue was detected by real-time quantitative PCR.2^stt PartSpecific circRNA target genes and their related proteins were identified by double luciferase reporter gene system and western imprinting technique.Adeno-associated virus?AAV?was injected into vivo to down-regulate or up-regulate the specific circRNA level of white adipose tissue.Statistical analysis was carried out by GraphPad Prism software.Results:1^stt Part1.1 Compared with the sedentary group?SE?,the weight gain of the exercise group?EX?mice was significantly reduced;Histomorphological observation showed that the volume of adipocytes in EX group was significantly reduced,and the number of adipocytes per unit area in adipose tissue was increased;immunohistochemical staining showed that UCP1 protein was expressed in the cell membrane.After treatment with UCP1 antibody,the EX group was treated with UCP1 antibody.The expression of genes related to browning in white adipose tissue of SE group and EX group was increased by immunohistochemical staining of UCP1 protein in cell membrane.The expression of UCP1,CIDEA,PPAR?,COX7a1,COX8b and PRDM16 in SE group and EX group had significant difference between the two groups?p<0.05?.The expression of UCP1 protein in SE group was higher than that in SE group,and the difference was statistically significant?p<0.05?.1.2 The results of exosome protein analysis showed that the exosomes of SE and EX groups had the expression of markers CD9,TSE101 and CD63 on their surfaces.1.3 After primary subcutaneous white adipocytes were co-incubated with exosomes extracted from SE mice?SE-exo?and with exosome extracted from EX mice?EX-exo?,the expression of PRDM16 and UCP1 in the latter increased significantly?p<0.05?.1.4 High throughput sequencing analysis of plasma exosome circRNA,SE and EX groups after plasma exosome sequencing differentially expressed circRNA,with |logFC|>2 and p value<0.05 as the critical value,EX group compared with SE group 124 up-regulated circRNA,134 down-regulated circRNA,further differentially expressed top 20 circRNA randomly selected 10 to be verified.The expression of circRNA was identical with that in the chip?p<0.05?.The circRNA with the highest expression difference was finally determined for further study.2^stt Part2.1 Bioinformatics analysis of microRNAs that interact with circRNA₀011487 was performed,and the microRNAs with the highest reliability were selected for verification.Fluorescence in situ hybridization of circRNA₀011487 and miR-328-3p showed that luciferase activity in NC group was significantly higher than that in co-transfection group of circRNA-WT and miR-328-3p mimics?p<0.05?,but there was no significant difference in Mut group.Double luciferase assay confirmed that the change of expression level of circRNA₀011487 affected the expression of miR-328-3p.Compared with negative control group,luciferase activity was significantly decreased in co-transfection group of PRDM16 and miR-328-3p mimics?P<0.05?.Luciferase activity was not significantly changed in co-transfection group of PRDM16-Mut.The changes of expression levels of circRNA₀011487 and miR-328-3p affected the expression of PRDM16 gene.After sh-circRNA₀011487 transfection,the expression of PRDM16 gene decreased significantly?p<0.05?.However,the inhibitor of miR-328-3p could reduce or inhibit the expression of PRDM16?p<0.05?;miR-328-3p mimics could make the expression of PRDM16 in cells significantly lower than that in the control group?p<0.05?;however,when miR-328-3p mimics and circRNA₀011487 overexpressed in cells,the expression of PRDM16 mRNA was significantly lower than that in the control group?p<0.05?.The expression of PRDM16 and UCP1 was not significantly lower in sh-circRNA₀011487 transfected group than in NC group?p<0.05?.There was no significant difference after co-transfection of miR-328-3p inhibitor.The expression of PRDM16 and UCP1 was significantly decreased by transfection of miR-328-3p mimics?p<0.05?,but there was no significant difference with co-transfection of circRNA₀011487 OE.2.2 After in vivo injection of AVV carrying sh-circRNA₀011487 sequence,the expression of circ₀011487 in white adipose tissue increased significantly and the expression of miR-328-3p decreased significantly?p<0.05?.Compared with SE group,the expression of UCP1,CIDEA,PPAR?,COX7a1 and COX8b in EX group was higher than that in SE group.The expression of UCP1 and PRDM16 increased significantly?p<0.05?,while the expression of PRDM16 and UCP1 decreased significantly in EX+sh-circ₀011487 group compared with EX+NC group?p<0.05?.CircRNA₀011487 can sponge with miR-328-3p in adipocyte,thus eliminating the inhibitory effect of miR-328-3p on PRDM16 and promoting the browning of white adipose tissue in mice.Conclusions:1 The expression of circRNA in exosome of mice after exercise training was different,which may be related to the effect of exercise.2 The combination of circRNA₀011487 and miR-328-3p inhibited the inhibitory effect of miR-328-3p on PRDM16,promoted the browning effect of fat and promoted the formation of beige cells,which provided a new idea for the effect of exercise on the body.