MiR-377Triggers Human Skin Fibroblasts Senescence By DNMT1Downregulation

Aims:We aim to identify the functions of DNA methyltransferase1(DMNT1) and the mechanisms of its regulation by the miR-377in the senescence of Human skin fibroblasts(HSF).Methods:1. Detecting the expression of DNMT1and Sirtl in replicative senescence model by Western blot after HSF replicative senescence model (n=8)(young group:P3-5,old group:P25-30) was set up. Afterwards the activity of the aging-related beta-galactosidase was measured via beta-galactosidase staining kit.2. Expression of DNMT1in HSF was knocked down by RNAi, and then Western blot was applied to detect the expression of DNMT1and Sirtl, furthermore the activity of beta-galactosidase was detected by beta-galactosidase staining kit.3.Based on the bioinformatics analysis of potential miRNA to bind to the3’UTR of DNMT1,we initially identified miR-377as an aging-related miRNA with potencial capacity to binding3’UTR of DNMT1.4.Real-time quantitative PCR was used to detect the expression of miR-377in replicative senescence model.5.Packaging miR-377over-expression lentivirus and an miR-377interference lentivirus.Transfected miR-377over-expression lentivirus to young human skin fibroblasts (P3)or miR-377interference Lentiviral to aging human skin fibroblasts, and identify its expression.Detecting the expression of DNMT1and sirt1and the activity of beta-galactosidase after transfection. 6.The sequence with WT or mutated miR-377binding site in3’UTR of DNMT1were cloned into the pRL-TK vector, respectively named pRL-TK-DNMT1-WT and pRL-TK-DNMT1-MUT, to study the transcriptional regulation of DNMT1by miR-377.7.To respectively build the epidermis and the skin fibroblasts normal aging model through collecting photophobic part of skins from people of different ages, and detect the expression of miR-377and DNMT1in vivo aging model.Results:1.In HSF replicative senescence model, compared to the younger group,the staining rate of beta-galactosidase is much higher in the aging groupMeanwhile,the expression of Sirtl remarkably decrease.2. The transfection of the effective DNMT1siRNA obtain a prominently increased beta-galactosidase staining rate of the treatment group and the notably lower expression of Sirtl.Thus,it is confirmed that the silence of DNMT1can induce senescence in vivo.3. Compared to the younger group,there is a significant augment of miR-377expression in the aging group.4. Overexpression of miR-377in young HSF results in a high staining rate of beta-galactosidase and increased expession of Sirtl, with statistical significance compared to the control group. The results were opposite when miR-377expression was knocked down in old HSF.5.An increased or lower expression of DNMT1can be induced by knockdown or overexpression of miR-377in young HSF respectively.6. luciferase reporter assay demostrates a direct interaction between miR-377and DNMT13’UTR.7. The high expression of miR-377but low of DNMT1are also validated in vivo.Concision:1.DNMT1plays a crucial role in senescence.2.miR-377can regulate the expression of DNMT1by directly targeting DNMT13’UTR and thus take essential roles in skin fibroblast senescence.