| Objective:To explore whether the aging vascular endothelial cells can regulate the aging process of skin fibroblasts through exosome-mediated microRNAs,and to further analyze the specific molecular mechanism of the aging vascular endothelial cells-derived exosome microRNAs regulating the fibroblasts senescnece.Methods:1.Firstly,the aging model of vascular endothelial cells induced by D-galactose was established.The expression of aging-related gene p53 was detected by beta-galactosidase staining and RT-q PCR.Then,the co-culture system of vascular endothelial cells and skin fibroblasts was established through Transwell chamber.After co-culture with aging vascular endothelial cells,the changes of skin fibroblasts biological functions were analyzed.The aging changes of skin fibroblasts after co-culture were analyzed by beta-galactosidase staining,the apoptosis was detected by Hoechst staining,and the proliferation function was analyzed by Ki67 staining.2.Firstly,according to the microarray analysis of aging-related microRNAs obtained by our research group,we analyzed the expression of aging-related microRNAs in senile vascular endothelial cells and skin fibroblasts cultured separately,and combined with the co-culture model of vascular endothelial cells and skin fibroblasts,we screened interesting microRNAs.Then,the expression of interesting microRNAs in endothelial cell exosomes was separated and identified by SBI exosome extraction kit,and the endothelial cell model of CD63-GFP expression was established by CD63-GFP lentivirus transfection.Combined with Cy3-specific microNA transfection technology,the endothelial cell-derived exosomes and related microRNAs were transfected.Tracer analysis was used to track the uptake effect of skin fibroblasts.At last,the exosome derived from young endothelial cells and the aging endothelial cells were extracted and co-culturewith skin fibroblasts directly.The aging changes of skin fibroblasts after co-culture were analyzed by beta-galactosidase staining,and the apoptosis was analyzed by Hoechst staining to further clarify the effect of aging endothelial cell exosome on skin fibroblasts.3.To establish the cell model of over-expression or low-expression of interesting microRNA,miR-326-3p and miR-767 mimics or inhibitors were transfected into skin fibroblasts by transfection of liposome,respectively.The expression of interesting microNAs in skin fibroblasts transfected by microRNA mimics or microRNA inhibitors was analyzed by RT-qPCR to determine the transfection effect of microRNA mimics or microRNA inhibitors.Then,the aging changes of skin fibroblasts after co-culture were analyzed by beta-galactosidase staining,the apoptosis was analyzed by Hoechst staining,the proliferation function was analyzed by Ki67 staining,and the biological function of skin fibroblasts transfected by specific microRNA mimic or microRNA inhibitor was analyzed.4.Up-regulate or down-regulate the expression of microRNA in primary skin fibroblasts,and detect the expression changes by RT-qPCR.Targetscan,a bioinformatics software,was used to predict the target genes of miR-326-3p and miR-767.RT-qPCR and Western blot were used to preliminarily analyze the correlation between interested microRNAs and target genes.Finally,the targeting relationship between miR-326-3p and target gene TLR4 and the targeting relationship between miR-767 and target gene TAB1 were identified by double luciferase reporter gene experiment.Result:1.In this study,the aging model of vascular endothelial cells was established by D-galactose induction.Compared with the control group,the number of positive cells and p53 gene expression increased significantly after 48 hours of aging induced by 20 g/L D-galactose(p<0.01).Then,the co-culture system of endothelial cells and skin fibroblasts was established through Transwell chamber.The co-culture group of non-aging vascular endothelial cells and skin fibroblasts(Control),aging vascular endothelial cells and skin fibroblasts(Model),aging vascular endothelial cells+GW4869 and skin fibroblasts(Model+GW)were observed respectively.After 48 hours of co-culture,compared with the control group,the number of aging-stained positive cells increased significantly in model group(p<0.01),the number of apoptotic-stained positive cells increased significantly(p<0.001),the number of Ki67 proliferation-stained positive cells decreased significantly(p<0.001);however,when aging endothelial cells were added with exosome inhibitor GW4869.There were no significant changes in senescence,apoptosis and number of positive cells for proliferation staining of fibroblasts in Model+GW group.2.The screening results of aging-related microRNAs showed that the expressions of miR-326-3p and miR-767 were significantly increased in senile vascular endothelial cells cultured alone(p<0.05),and the expressions of miR-326-3p and miR-767 were significantly decreased in senile skin fibroblasts cultured alone(p<0.05).However,in the co-culture system of vascular endothelial cells and skin fibroblasts,the expressions of miR-326-3p and miR-767 in skin fibroblasts in Model group were significantly higher than those in control group.Then,the exosomes in endothelial cell culture medium were isolated and identified.Under ultramicro-electron microscopy,the "saucer-like"vesicles in the extract were observed,with a diameter of about 60 nm.Nano particle size analysis showed that the diameter of the extract was mainly concentrated in the range of 40 to 100 nm.Then,after extracting the exosomes of the endothelial cell culture medium of the non-aging vascular endothelial cells and the aging vascular endothelial cells cultured directly intervene in skin fibroblasts,compared with the exosomes of the non-aging vascular endothelial cells(EXO-control,Exo-c),the levels of miR-326-3p and micR-767 in the exosomes of the aging vascular endothelial cells(Exo-s)also increased significantly.Finally,CY3-miR-326-3p was transfected into endothelial cells labeled with GFP-CD63.The results of tracer analysis showed that both green fluorescence of GFP-CD63 and red fluorescence of miR-326-3p appeared in co-cultured skin fibroblasts.More interestingly,the addition of exosomes from non-senile vascular endothelial cells as control group(Exo-c)and exosomes from senile vascular endothelial cells(Exo-s)not only increased the number of senile positive cells in skin fibroblasts(p<0.001),but also promoted the staining of apoptotic cells in skin fibroblasts(p<0.001),suggesting that endothelial cells are accessible.MicroRNAs mediate the aging process of skin fibroblasts through exosome pathway.3.Transfection with miR-326-3p mimic or miR-326-3p inhibitor can significantly increase or decrease the expression of miR-326-3p in skin fibroblasts.The senescence of skin fibroblasts was analyzed by beta-galactosidase staining.Compared with the negative control group(NC-mimic),the number of senescence-positive cells in skin fibroblasts transfected with miR-326-3p mimic increased significantly(p<0.01);compared with the negative control group(NC-inhibitor),the number of senescence-positive cells in skin fibroblasts transfected with miR-326-3p inhibitor decreased significantly(p<0.05).Hoechst apoptotic staining was used to analyze the apoptosis of skin fibroblasts.Compared with the negative control group(NC-mimic),the number of apoptotic staining positive cells of skin fibroblasts transfected with miR-326-3p mimic increased significantly(p<0.01);compared with the negative control group(NC-inhibitor),the number of apoptotic staining positive cells of skin fibroblasts transfected with miR-326-3p inhibitor decreased significantly(p<0.05).Ki67 proliferation assay kit was used to analyze the cell proliferation function.Compared with the control group(NC-mimic),the number of Ki67 proliferation staining cells in skin fibroblasts transfected with miR-326-3p mimic decreased significantly(p<0.001);compared with the control group(NC-inhibitor),the number of Ki67 proliferation cells in skin fibroblasts transfected with miR-326-3p inhibitor did not change significantly.After transfection with miR-767 mimic or miR-767 inhibitor,the expression of miR-767 in skin fibroblasts could be significantly increased or decreased.The aging of skin fibroblasts was analyzed by beta-galactosidase aging staining.Compared with the control group(NC-mimic),the number of aging positive cells in skin fibroblasts transfected with miR-767 mimic increased significantly(p<0.05);compared with the control group(NC-inhibitor),the number of aging positive cells in skin fibroblasts transfected with miR-767 inhibitor decreased significantly(p<0.05).Hoechst apoptotic staining was used to analyze the apoptotic status of skin fibroblasts.The results showed that the number of apoptotic staining positive cells of skin fibroblasts transfected with miR-767 mimic increased significantly(p<0.001)compared with the control group(NC-mimic),and the number of apoptotic staining positive cells of skin fibroblasts transfected with miR-767 inhibitor decreased significantly(p<0.01).Ki67 proliferation assay kit was used to analyze the cell proliferation function.Compared with the control group(NC-mimic),the number of Ki67 proliferation staining cells in skin fibroblasts transfected with miR-767 mimic decreased significantly(p<0.001);compared with the control group(NC-inhibitor),the number of Ki67 proliferation cells in skin fibroblasts transfected with miR-767 inhibitor did not change significantly.4.Targetscan was used to predict the target genes of miR-326-3p and miR-767.The results showed that miR-326-3p could bind to 3'-UTR of TLR4 and miR-767 could bind to 3'-UTR of TAB1.Compared with the control group(NC-mimic),the expression of TLR4 gene in skin fibroblasts transfected with miR-326-3p mimic decreased significantly(p<0.05)and the expression of TLR4 protein was decreased.;however,compared with the control group(NC-inhibitor),the expression of TLR4 gene in skin fibroblasts transfected with miR-326-3p inhibitor increased significantly(p<0.05)and the expression of TLR4 protein was increased.Compared with the control group(NC-mimic),the expression of TAB1 gene in skin fibroblasts transfected with miR-767 mimic decreased significantly(p<0.001)and the expression of TAB1 protein was decreased.;however,compared with the control group(NC-inhibitor),the expression of TAB1 gene in skin fibroblasts transfected with miR-767 inhibitor increased significantly(p<0.001)and the expression of TAB1 protein was increased.In addition,the double luciferase reporting test showed that the fluorescence value of miR-326-3p mimic/TLR4 WT co-transfer group was significantly lower than that of control group,but there was no significant difference between miR-326-3p mimic/TLR4 MUT co-transfer group and control group.It is suggested that TLR4 is the direct target gene of miR-326-3p.The fluorescence value of miR-767 mimic/TAB1 WT co-transfer group was significantly lower than that of control group,but there was no significant difference between miR-767 mimic/TAB1 MUT co-staining group and control group,suggesting that T AB1 is the direct target gene of miR-767.Conclusion:1.Through the co-culture system of vascular endothelial cells and skin fibroblasts,we found that vascular endothelial cells may regulate the aging process of skin fibroblasts through exosome pathway.2.MiR-326-3p and miR-767 in exosomes of senile endothelial cells,can accelerate the senescence of skin fibroblasts,promote cell apoptosis,inhibit cell proliferation and can be absorbed by skin fibroblasts,and regulate the biological function of cells.3.MiR-326-3p and miR-767 can regulate the apoptosis and proliferation of skin fibroblasts and accelerate the aging of skin fibroblasts.4.This study confirms that miRNA-326-3p can directly inhibit the expression of TLR4 target gene,and miR-767 can directly inhibit the expression of TAB 1 target gene. |
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