| Objective: Atopic dermatitis(AD)is a common and highly heterogeneous inflammatory dermatitis,which is characterized by pruritus,dry skin,and eczema-like rash.Recent data shows that up to 20% of the children and up to 3% of the adults are affected by AD in the world.Besides,the prevalence rate is still gradually increasing.As a systemic disorder,the clinical presentation of AD is characterized by pruritus and eczematous,the pathology of AD is characterized by skin barrier defects and immune disorders.Recent evidences also suggest that AD is associated with asthma,atopic rhinitis and other atopy or allergic diseases.Although glucocorticoids,neurophosphatase inhibitors,antihistamines,antibiotics,cyclosporine,tripterygium wilfordii,ultraviolet therapy and traditional Chinese medicine,especially the biological agent dupilumab,can effectively attenuate AD symptoms,all of them can only suppress the inflammation and do not completely cure the disease.Genetic studies on AD have identified some genes are associated with the regulation of the epidermal barrier or innate immune response.Therefore,it is desirable to prevent and treat AD by detecting and regulating the expression of target genes related to pathogenesis of AD.Keratinocyte(KC)cells are found in the deepest basal layer of the stratified epithelium that comprises the epidermis,and are account for more than 95% of the epidermal cells.KCs are maintained at various stages of differentiation in the epidermis and are responsible for forming tight junctions with the nerves of the skin.Moreover,it's well known that skin is the first line of defense and KCs serve as a barrier between an organism and its environment.KCs also serve a chemical immune role as immunomodulators,responsible for secreting inhibitory cytokines in the absence of injury and stimulating inflammation and activating Langerhans cells in response to injury.Several studies revealed that the excessive cell proliferation of KCs was associated with the development and progression of AD.HaCaT cells are an immortalized cell lines derived from normal adult skin KC,which was the commonly used in the study of skin in vitro.Micro-RNA(micro RNA,miRNA)are a class of endogenous non-coding RNA,with a length of about 18-22 nucleotides,which perform biological functions mainly by targeting target genes to degrade mRNA or block translation.A large number of studies have shown that miRNA is related to the occurrence and development of immune and inflammatory diseases,including AD.Numerous studies demonstrated that peripheral blood miRNA expression profile has becoming a disease-related molecular tag.Although studies have shown that miR-375 has a low expression in the skin of patients with AD,the detailed mechanism of miR-375 in keratinocytes is not clear.Lympho-epithelial Kazal type inhibitor(LEKTI)was significantly down-regulated in KCs of patients with AD.LEKTI could inhibit the activity of Kallikrein(KLK)-5 and Kallikrein-7,and the deletion of LEKTI will lead to the peeling of stratum corneum and thinning of epidermis,the decrease of filaggrin(FLG)level and the increase of interleukin-1?(IL-1?),protease activated receptors-2(PAR2)and intercellular adhesion molecule-1(ICAM1).Yes-associated protein 1(YAP1)could regulate the proliferation of epidermal stem cells,and YAP1 overexpression lead to the decrease of LEKTI and the immortalization of normal keratinocytes.Some studies have shown that YAP1 is one of the targets of miR-375,but it is not known whether miR-375 could increase the level of LEKTI by down-regulating YAP1.Methods: 1.Cell culture: Human HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5%CO2 in a humidified incubator.2.miR-375-3p mimics and miR-375-3p inhibitor transfection: HaCaT cells at logarithmic growth phases were cultured in DMEM medium containing 10% fetal bovine serum for 24 h,followed by incubation in DMEM without fetal bovine serum for 1 h.50 pmol miR-375-3p mimic,miRNA mimics negative control(mimics NC),and miR-375-3p inhibitor negative control(inhibitor NC)were transfected into the cultured HaCaT cells using Lipofectamine2000 for 4 h incubation following the manufacturer's instructions,then the medium was discarded and replaced by DMEM containing 10% fetal bovine serum.3.Establish of HaCaT cells inflammation model: HaCaT cells were co-cultured with medium containing TNF-?(10 ng/ml)and IFN-?(10 ng/ml)for 6 h.4.Transfection of YAP1 overexpression plasmid: 2 ?g YAP1 overexpression plasmid and 2 ?g empty plasmid were transfected into HaCaT cells with liposome 2000 for 4h.then the medium was discarded and replaced by DMEM containing 10% fetal bovine serum.After incubation for 48 h,the expression levels of YAP1 in transfected cells were detected by Western blotting.5.The viability of HaCaT cells was detected by CCK-8.6.q RT-PCR and western blotting were used to detect the changes of mRNA and protein levels of the target gene.7.The levels of IL-1? and IL-IL-6 secretion in HaCaT were detected using ELISA kit.8.The targeting relationship between miR-375-3p and YAP1 was confirmed through dual luciferase reported system.Results: 1.Overexpression of miR-375-3p could effectively inhibit the inflammatory proliferation of HaCaT cells-induced by TNF-? and IFN-?.CCK-8 assay results showed that the viability of HaCaT cells in the model group was significantly higher than that in the normal control group(P<0.05).In contrast,compared with model group,overexpression of miR-375-3p inhibit cell viability of HaCaT cells,while miR-375-3p knockdown increased cell viability(P<0.05).2.Overexpression of miR-375-3p reduced the secretion of IL-1? and IL-6,as well as the activation of nuclear factors(NF-?B)-induced by TNF-? and IFN-?.Compared with the control group,TNF-? and IFN-? treatment led to the increased release of IL-1?and IL-6(P<0.01),NF-?B translocation from cytoplasm to nuclear(P<0.01).In contrast,overexpression of miR-375-3p rescued the elevation of IL-1? and IL-6(P<0.01),and blocked the activation of NF-?B(P<0.01),while miR-375-3p knockdown exerted opposite effects.3.Overexpression of YAP1 promotes the secretion of HaCaT cytokines(IL-1? and IL-6)and the activation of nuclear factors(NF-?B)induced by TNF-? and IFN-?.Compared with the model group,overexpression of YAP1 could significantly promote the proliferation of HaCaT cells(P<0.01),increase the secretion of IL-1? and IL-6(P<0.01),increase the nuclear transfer of NF-?B(P< 0.01)and decrease the expression of LEKTI(P<0.01).4.YAP1 Serves as a direct target gene of miR-375-3p: Target Scan predict results show that YAP1 3'UTR has a potential binding site for miR-375-3p.Moreover,The results of double luciferase reporter system showed that the luciferase activity was significantly decreased in cotransfection miR-375-3p mimics and wild-type YAP13'UTR reporter vector group,but there was no significant change in cotransfection miR-375-3p mimics and mutant YAP1 3'UTR group(P<0.05),indicating that miR-375-3p targeted YAP1 expression by specifically binding its 3'-UTR.Compared with the control group,the levels of YAP1 mRNA and protein in the model group were increased significantly,while the level of LEKTI protein decreased(P< 0.01).Compared with the model group,the overexpression of miR-375-3p significantly decreased the level of YAP1 at mRNA and protein levels(P<0.01).5.Overexpression of YAP1 inhibited the decrease of IL-1? and IL-6 levels and NF-?B inactivation-induced by miR-375-3p mimics.Compared with miR-375-3p mimics transfection group,the levels of IL-1? and IL-6,p-I-?B expression and NF-?B nuclear translocation in cells co-transfected with YAP1 overexpression vector and miR-375-3p mimics were significantly increased(P<0.01).Conclusions: 1.Overexpression of miR-375-3p could inhibit the inflammatory proliferation,secretion of inflammatory factors,and NF-?B activation-induced by TNF-? and IFN-?.2.YAP1 overexpression significantly promotes inflammatory factor release and NF-?B nuclear translocation.3.YAP1 serves as a direct target gene of miR-375-3p,miR-375-3p could inhibit YAP1 protein expression by binding to its3'-UTR,and thereby promoting LEKTI protein expression. |
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