Reduction Of MiR-133a-3p Contributes To Apoptosis And Gasdermin E-mediated Pyroptosis Of Keratinocytes In Skin Exposed To Ultraviolet B Radiation

Background and objectivesSeveral skin diseases are closely associated with ultraviolet(UV)radiation.Excessive UV radiation induces acute inflammatory skin reactions.such as sunburn.After UVB radiation,tissue damage is usually concentrated in the epidermis,composed primarily of keratinocytes,due to the low penetration power of UVB.Cell death is a key process in keratinocytes exposed to UVB radiation,of which apoptosis has long been the only form of regulated cell death found.Recent studies including our group have shown that UVB radiation can induce GSDME-mediated pyroptosis in keratinocytes.Our preliminary study also observed topical metformin had a protective effect on keratinocyte death in UVB-induced skin damage.It is not clear,however,whether metformin protects against different types of UVB-induced regulated cell death.It has been shown that miR-133a-3p is involved in the pathogenesis of inflammatory skin diseases(e.g.,psoriasis,atopic dermatitis),while miR-133a-3p has also been found to be involved in processes such as apoptosis,GSDMD-mediated pyroptosis.However,it is still unclear whether miR-133a-3p co-regulates both apoptosis and pyroptosis in keratinocytes after UVB irradiation.The aim of this study was to clarify the differential expression of miR-133a-3p in keratinocytes after UVB radiation,as well as to investigate how miR-133a-3p regulates apoptosis and GSDME-mediated pyroptosis and the mechanism involved.Materials and methods1.We established UVB radiation-induced acute photodamage model in mice and used topical metformin as treatment.miR-133a-3p level was detected by real-time fluorescence quantitative PCR(qPCR).Level and localization of miR-133a-3p was detected by RNA in situ hybridization.Proteins of apoptosis and GSDME-mediated pyroptosis were detected in the epidermis by Western blot(WB).Level and localization of cleaved caspase-3 and cleaved PARP were detected by immunofluorescence.2.We injected agomir miR-133a-3p intradermally into UVB-radiated mice.Epidermal cell death level was detected by TUNEL staining.Proteins of apoptosis and GSDME-mediated pyroptosis in the epidermis were detected by WB.3.We established UVB radiation-induced HaCaT cell death model and added metformin to inhibit cell death.Cell death level was detected by Trypan blue staining.Proteins of apoptosis and GSDME-mediated pyroptosis were detected by WB.miR-133a-3p levels were detected by qPCR.Adhesion level of THP-1 cell co-cultured with HaCaT cells was detected.4.HaCaT cells were transfected with miR-133a-3p mimic or inhibitor to explore the effect of miR-133a-3p on UVB radiation-induced HaCaT cell death.Metformin was added to UVB-radiated HaCaT cells transfected with miR-133a-3p inhibitor.Proteins of apoptosis and GSDME-mediated pyroptosis in the above systems were detected by WB.5.Possible targets of miR-133a-3p were predicted by bioinformatics,and dual luciferase reporter gene assay was to verify the target mRNA CYLD.CYLD was detected by qPCR and WB after altering miR-133a-3p levels.In UVB-radiated mice with agomir miR-133a3p injection,CYLD in the epidermis was measured by WB.Transduction of shRNAcontaining lentiviral in HaCaT cells to knock down CYLD,as well as transfection of miR133a-3p inhibitor in CYLD knockdown HaCaT cells were to verify whether CYLD is involved in the protective effect of miR-133a-3p against UVB-radiation induced cell death.Proteins of apoptosis and GSDME-mediated pyroptosis in the above systems were detected by WB.Results1.UVB radiation induced acute skin damage in mice,as well as apoptosis and GSDMEmediated pyroptosis in keratinocytes,which were inhibited after topical metformin treatment.miR-133a-3p levels in the epidermis were reduced after UVB radiation and restored after metformin treatment.2.Intradermal injection of agomir miR-133a-3p alleviated mice skin damage induced by UVB radiation,reduced the overall level of keratinocyte death,and inhibited apoptosis and GSDME-mediated pyroptosis in keratinocytes.3.UVB radiation induced apoptosis and GSDME-mediated pyroptosis in HaCaT cells,accompanied by decrease of miR-133a-3p in HaCaT cells,and also induced adherence of THP-1 cells co-cultured with HaCaT cells.These effects were suppressed after metformin treatment.4.Over-expression or inhibition of miR-133a-3p inhibited or promoted UVB radiationinduced apoptosis and GSDME-mediated pyroptosis in HaCaT cells,respectively.The protective effect of metformin against UVB-induced apoptosis and GSDME-mediated pyroptosis was suppressed in miR-133a-3p-inhibited HaCaT cell.5.The 3’-UTR region of CYLD binds to miR-133a-3p.miR-133a-3p over-expression or inhibition reduces or promotes C YLD expression in HaCaT cells,respectively.Intradermal injection of agomir miR-133a-3p reduced the expression of epidermal CYLD.The exacerbating effect of miR-133a-3p inhibition on UVB-induced apoptosis and GSDMEmediated pyroptosis was suppressed in CYLD knockdown HaCaT cells.Conclusions1.miR-133a-3p level is reduced in the epidermis of UVB-radiated skin,and restoration of miR-133a-3p level can alleviate UVB-induced skin damage.2.In UVB-induced skin damage,miR-133a-3p inhibited both apoptosis and GSDMEmediated pyroptosis in keratinocytes and was involved in the protective effect of metformin on keratinocyte death.3.CYLD,one of the target molecules of miR-133a-3p,is involved in the protective effect of miR-133a-3p in inhibiting UVB-induced keratinocyte apoptosis and GSDME-mediated pyroptosis.