| Objective: micro RNAs(miRNAs)play a variety of important regulatory roles in cells and play an important role in the aging process by regulating gene expression.Previous studies of our group have shown that the expression of miR-326-3p in vascular endothelial cell-derived exosomes is increased during the aging process,and it can accelerate the aging of skin fibroblasts.However,the mechanism of miR-326-3p’s targeting regulation of TLR4 on cell senescence has not been clarified.This study will deeply analyze the biological functions of miR-326-3p in skin fibroblasts and vascular endothelial cells,analyze the role of low expression of TLR4 in promoting cell aging,and explore the correlation between miR-326-3p and TLR4 and the skin of aging mice in vivo.The molecular mechanism of miR-326-3p targeting TLR4 regulating skin fibroblast senescence was further verified in this study.Methods:1.Previous studies of our group have confirmed that miR-326-3p significantly increased in aging vascular endothelial cells.In order to investigate the effect of miR-326-3p on the aging of vascular endothelial cells,this study transfected miR-326-3p mimic(overexpression)/inhibitor(low expression)into the vascular endothelial cells.The expression of miR-326-3p was monitored by q RT-PCR.The impact of miR-326-3p on the aging,proliferation and apoptosis of vascular endothelial cells were analyzed by β-galactosidase staining,Ki67 proliferation detection and Hoechst apoptosis staining.2.Our previous study confirmed that miR-326-3p overexpression can promote the aging of skin fibroblasts.In order to investigate the intervention effect of low expression of miR-326-3p on aging skin fibroblasts,miR-326-3p inhibitor was transfected into D-gal-induced aging skin fibroblasts.β-galactosidase staining,Ki67 proliferation test and Hoechst apoptosis staining,was used to analyzing the function changes in the cells,and the intervention of miR-326-3p with low expression on the cell function of skin fibroblasts,including senescence,proliferation and apoptosis,was analyzed by rescue experiment.3.The research group has previously confirmed that TLR4 is the directly target gene of miR-326-3p.So as to research the effect of TLR4 on the biological function of cells,the correlation between miR-326-3p and TLR4 gene expression in skin fibroblasts was verified by q RT-PCR after transfection with miRNA mimic/inhibitor.The correlation of TLR4 protein expression in cells was monitored by WB.So as to research the effect of TLR4 on cell senescence,siRNA-TLR4 was transfected into skin fibroblasts and vascular endothelial cells by liposome transient transfection method,respectively.β-galactosidase staining,Ki67 proliferation detection,Hoechst apoptosis staining and flow cytometry were performed.The effects of low expression of TLR4 on senescence,proliferation,apoptosis and cell activity of skin fibroblasts and vascular endothelial cells were analyzed.4.In order to investigate the expression of miR-326-3p and TLR4 in aging skin tissue in vivo,we established an aging animal model of D-galactose,and observed the morphological changes of aging skin tissue by HE staining and Masson tricolor staining.The differential expression of miR-326-3p and TLR4 gene expression in skin tissues were verified by q RT-PCR.The protein expression of p21 and TLR4 in tissues was detected by Western Blot.Results:1.For the sake of analyzing the biological function of miR-326-3p in vascular endothelial cells,miR-326-3p mimic and miR-326-3p inhibitor were used to intervene vascular endothelial cells and D-gal-induced aging vascular endothelial cells respectively.The q RT-PCR analysis results showed that,after the intervention of miR-326-3p mimic,the expression of miR-326-3p in vascular endothelial cells was remarkably increased(P<0.01),and after the intervention of miR-326-3p inhibitor,the expression of miR-326-3p in vascular endothelial cells was remarkably decreased(P<0.05).Compared with NC-mimic group,transfection of miR-326-3p mimic in vascular endothelial cells remarkably increased the positive rate of senescence staining and apoptosis staining(P<0.01),but also remarkably decreased the positive rate of proliferative staining(P<0.01).Compared with NC-inhibitor+D-gal group,miR-326-3p inhibitor+D-gal group not only remarkably decreased the positive rate of aging and apoptosis staining cells of vascular endothelial cells(P<0.01).It also resulted in a remarkably increase in the positive rate of proliferative staining cells(P<0.01).2.To analyze the intervention effect of miR-326-3p low expression on skin fibroblast senescence,the cells were randomly divided into CTRL,NC,NC+D-gal and miR-326-3p inhibitor+D-gal group.NC inhibitor and miR-326-3p inhibitor were transfected into D-Gal-treated skin fibroblasts,respectively.The results showed that there was no statistical significance between CTRL group and NC group(P > 0.05).Compared with the NC group,the positive cell rate of senescence staining and apoptotic staining were increased in the NC+D-gal group(P<0.001),while the positive cell rate of proliferation staining was remarkably decreased(P<0.001).In comparison with the NC+D-gal group,miR-326-3p inhibitor+D-gal group remarkably decreased the expression of senescence staining positive rate of cells(P<0.01)and apoptosis staining positive rate of cells(P<0.001),while proliferation staining positive rate of cells was remarkably increased(P<0.001).In the rescue experiment,it was found that miR-326-3p inhibitor+D-gal group could inhibit cell senescence and apoptosis induced by D-gal,and promote cell proliferation and cell activity,which further verified that the low expression of miR-326-3p could delay the senescence of skin fibroblasts.3.In order to further explore the molecular mechanism of TLR4 in the aging process,the low-expression TLR4 cell model was established by siRNA to further verify the effect of target gene TLR4 on senescence related functions.miR-326-3P mimic/inhibitor was transfected into skin fibroblasts and vascular endothelial cells,respectively.The results showed that,In comparison with the CTRL group,siRNA-TLR4-1#,siRNA-TLR4-2# and siRNA-TLR4-3# groups could not merely increase the expression of senescence staining positive rate and apoptosis staining positive rate,but also decrease the expression of proliferation staining positive rate and cell survival positive rate.In comparison with the D-gal group,D-gal group and siRNA-TLR4-1#,siRNA-TLR4-2# and siRNA-TLR4-3# groups could diminishing the expression of senescence staining positive cell rate and apoptosis staining positive cell rate,and also increase the expression of proliferation staining positive cell rate,in which the siRNA-TLR4-2# group promoted cell and apoptosis.The inhibition of cell proliferation was most obvious.The rescue experiment showed that in comparison with the NC group,NC+D-gal group,siRNA-TLR4-2# group,miR-326-3p inhibitor+D-gal group and miR-326-3p inhibitor+siRNA-TLR4-2# group all increased the expression of senile staining positive cell rate of skin fibroblasts(P<0.001).However,in comparison with the D-gal+miR-326-3p inhibitor group,the expression of positive cell rate of aging staining of skin fibroblasts in D-gal+miR-326-3p inhibitor+siRNA-TLR4-2# group decreased(P<0.001).4.In order to further analyze the molecular mechanism of miR-326-3p regulating skin aging in vivo,D-galactose animal aging model was established.The results indicate that the expression levels of p21 m RNA and protein in the skin tissues of mice induced by D-galactose for 3 months were significantly higher than those in the CTRL group,suggesting that D-galactose induced animal aging model was successfully established.The results indicate that HE staining and Masson three-color staining,the aging skin tissue structure had obvious pathological changes.In the CTRL group,the skin tissue structure was relatively intact,the epidermis was thickened and the collagen fibers in the dermis were increased.However,in the Aging group,the skin tissue structure was relatively damaged,the epidermis became thinner and the collagen fibers in the dermis decreased.The q RT-PCR analysis showed that the expression of miR-326-3p in the Aging group was visibly increased(P<0.01),whereas the expression of TLR4 m RNA was significantly decreased(P<0.01),at the same time,in comparison with the CRTL group.WB analysis indicated that in comparison with the CRTL group,the expression of TLR4 protein in the Aging group was significantly down-regulated(P<0.01).Conclusion: Our experimental results further confirmed that the overexpression of miR-326-3p can lead to aging of vascular endothelial cells,meanwhile,the low expression of miR-326-3p can also postone the aging of skin fibroblasts.In addition,TLR4 as the target gene of the low expression of miR-326-3p can accelerate the aging of vascular endothelial cells and skin fibroblasts,and the low expression of miR-326-3p can delay the aging process of skin fibroblasts by up-regulating the expression of TLR4.Meanwhile,in vivo experiments further confirmed that the high progress of miR-326-3p may promote the aging of skin tissue by inhibiting TLR4.This study revealed the molecular mechanism of miR-326-3p regulating cell aging,suggesting that miR-326-3p can be used as a molecular target against skin aging and has potential pharmacological application value. |
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