| Aging is a complex process including different systems and organs which has a series of specific changes. Skin aging whose nature and basis is skin cell senescence is the direct manifestation of organism aging. Skin cell senescence is not only controlled by genetics but also regulated by epigenetics. Epigenetics is an important mechanism of senescence, including DNA methylation, histone modification, chromatin remodeling, miRNA regulation and so on.MiRNA-217is a micro non-coding RNA which is highly expressed in human umbilical vein endothelial cells and coronary artery endothelial cells. It has been proved that miR-217can target regulate KRAS to inhibit tumorigenesis in the pancreatic ductal adenocarcinoma and can target regulate Runx2to affect osteocyte maturity. Some researches reveal miR-217increases in the senescent human vascular endothelial cells (HVEC) and can induce senescence of HVEC by inhibiting sirtl gene. However, so far, the function of miR-217in the mechanism of aging remains to be defined. Our previous studies by microRNA microarray have shown that miR-217is increased in the senescent human skin fibroblasts (HSF) compared with the young HSF. So we speculate that miR-217may play an important role in the senescence of HSF.To prove the hypothesis, we used the three parts following:Firstly, detected and compared the expression of miR-217in the young and senescent HSF. Secondly, to investigate the role of miR-217in the HSF senescence, overexpressed miR-217in the young HSF and detected the senescent phenotype, expressions of related senescent protein. Thirdly, to investigate the mechanism of miR-217in the senescence of HSF:(1) through bioinformatic prediction, discovered miR-217may target regulate DNA methyltransferase1(DNMT1) and proved the correlation between miR-217and DNMT1through Dual Luciferase Reporter System;(2) detected the regulation of miR-217to the expression of DNMT1in HSF and to the senescence process in HSF.In our study, we found miR-217could induce the senescence of HSF through inhibiting DNMT1. Taken together, our findings can provide new directions in research and therapy of skin aging.Part I Expression of miR-217in young and senescent HSFObjective:To investigate the expression of miR-217in young and senescent of HSF.Methods:Gathered the skin of healthy teenagers (<10years old), healthy middle-aged (30-50years old) and healthy elderly group (>60years old). Primary cultured HSF, passaged the HSF of teenagers (young cells:P3-5, senescent cells:P20-25), and detected the expression of miR-217by Real-time PCR.Results:Compared with teenager group, expression of miR-217was significantly increased both in middle-aged and elderly group (middle-aged:P<0.05, elderly group:P<0.01), and miR-217in elderly group also increased compared with middle-aged group (P<0.01). In the replicative senescence model, miR-217in the senescent HSF was increased compared with young HSF (P<0.01).Conclusion:miR-217expression was significantly increased in elderly HSF and senescent HSF. Part Ⅱ Relationship between the overexpression of miR-217and HSF senescenceObjective:To investigate the role of miR-217in the HSF senescenceMethods:HSF of teenager group was transfected by hsa-miR-217lentivirus or negative control virus.48h later, the expression of miR-217was detected by Real-time PCR. The proliferative capacity was tested by methyl thiazoly tetrazolium (MTT) assay, the percentage of positive cells was detected by SA-β-gal staining, and the expression of senescence-related proteins P53, P21and sirt1were detected by westen blot.Results:After transfected by hsa-miR-217lentivirus, the expression of miR-217increased significantly(P<0.01), the proflieration of cells was obviously inhibited(P<0.05), the percentage of SA-β-gal staining positive cells increased (P<0.01), and the expression of senescence-related proteins P53and P21were upregulated, while sirt1was downregulated, respectively (P<0.05)Conclusion:The overexpression of miR-217could induce the senescence of HSF, and miR-217played an important role during this process. Part Ⅲ miR-217regulated DNMT1in HSF directly and affected the senescence processObjective:To investigate the mechanism of miR-217in the senescence of HSFMethods:MICRORNA.ORG website was used to predict whether DNMT1was the targeting gene of hsa-miR-217, and the wildtype and mutation of DNMT1-3’UTR luciferase reporter recombinant plasmids were constructed.293T cells were transfected by these plasmids respectively, and4h later, cells were transfected by miR-217mimics, then48h later, the relative luciferase activities were analyzed by Dual Luciferase Reporter Gene Assay System (DLR). DNMT1-shRNA lentivirus was transfected into the young HSF and detected the senescence phenotype after48h. Stably transfected HSF cells of teenager group from Part Ⅱ were used to test the expression of DNMT1by westen blot, and after these cells were transfected by DNMT1-cDNA lentivirus, the percentage of positive cells was detected by SA-β-gal staining.Results:Through bioinformatic prediction, we discovered miR-217may targeted regulate DNMT1. The results of DLR showed that when overexpressing miR-217, the luciferase activity of wildtype DNMT1-3’UTR transfected cells decreased than control group (P<0.05), but no obvious change was found in mutation group. After inhibiting DNMT1in the young HSF, the proflieration of cells was obviously inhibited (P<0.05), the percentage of SA-β-gal staining positive cells increased (P<0.01). After overexpressing miR-217in young HSF of teenager group, western blot showed the expression of DNMT1decreased significantly (P<0.01). After transfected by hsa-miR-217shRNA and DNMT1-shRNA lentivirus, the percentage of positive cells increased (P0<.05)Conclusion:DNMT1was the targeting gene of miR-217, and miR-217could targeting combine the3’UTR region of DNMT1. The expression of DNMT1decreased in the replicative senescent HSF, and inhibiting DNMT1could induce the senescence of HSF. miR-217regulated the senescence of HSF by modulating the expression of DNMT1. |
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