Methyltransferase METTL14 Affects Skin Fibroblasts Photoaging Via N6-Methyladenosine-Dependent Regulation Of MiR-100-3p

BackgroundSkin aging is the cumulative effect of various internal and external factors,including intrinsic aging caused by genetic factors and extrinsic aging caused by environmental factors.Long-term repeated exposure to ultraviolet radiation is the main factor of extrinsic aging,which is also called photoaging.With the extension of life expectancy,the incidence of photoaging-related tumors is also increasing,such as basal cell carcinoma,squamous cell carcinoma,etc.Furthermore,as the demand for cosmetology grows,the prevention and treatment of skin photoaging becomes increasingly crucial.In recent years,research on RNA epigenetics has steadily evolved in a variety of domains,with RNA m6A(N6methyladenosine)modification,as the most prevalent and prolific RNA alteration in eukaryotic cells,gradually emerging as a new research hotspot.It is found in both messenger RNA(mRNA)and non-coding RNA(ncRNA).A few studies have found that m6A is related to UV radiation.MicroRNA(miRNA),the most researched ncRNA,is also becoming increasingly significant in photoaging.However,the research on miRNA-related m6A modification is still rare,and there is no related report in the field of skin photoaging.Therefore,this study aimed to explore the mechanism of miRNA m6A modification in skin photoaging.Methods1.Induce skin fibroblast photoaging with single UVB(30mJ/cm2)and validate the model with aging-related β-galactosidase staining,CCK-8 cell viability assay,flow cytometry,and Western Blot of aging markers(p53,p21and type Ⅰ collagen).2.Determine the m6A modification level of total RNA in photoaged cells by m6A Dot Blot test.Detect m6A-regulated genes using RT-qPCR and Western Blot.Use lentivirus to overexpress or knock down the genes identified above,and then assess their effect on photoaging phenotypes.3.Use miRNA high-throughput sequencing and RT-qPCR to determine the differentially expressed miRNAs in photoaged cells,and validate the level of m6A on pri-miRNA by RIPqPCR and MeRIP-qPCR.4.Determine the downstream target mRNAs of miRNA through miRNA-mRNA interaction database,GSE119009 dataset,RT-qPCR,Western Blot,and dual luciferase reporter gene assays.5.Explore the effect on skin fibroblasts photoaging phenotype by transfecting miRNA mimic/inhibitor and target gene silencing or overexpression plasmids.Results1.After 24 hours of single UVB(30mJ/cm2)exposure,cell volume rose,flattened,and refractive index decreased.Cell viability reduced,and the proportion of aging-relatedgalactosidase-positive cells increased.The aging marker type I collagen was significantly reduced,while p5 3 and p21 were significantly increased.Cells were blocked in G1,meaning that the proportion of cells in G1 in the UVB group was significantly increased,while the proportion of cells in G2 and S was significantly decreased.2.The m6A modification level of total RNA decreased in photoaged cells,and the expression of METTL14 mRNA and protein levels also decreased.Overexpression of METTL14 alleviated the photoaging impact caused by UVB,implying that METTL14 overexpression exerted a photoprotective function.3.High-throughput sequencing revealed 41 down-regulated miRNAs in photoaged cells(P<0.05,log2(Fc)≥1.5)and RT-qPCR confirmed miR-100-3p was significantly reduced.The expression of miR-100-3p increased following METTL14 overexpression,while the level of its precursor pri-miR-100 declined.RIP-qPCR data revealed that the expression levels of pri-miR-100 bound to DGCR8 were significantly increased in METTL14overexpressing cells.MeRIP-qPCR data revealed that the expression levels of pri-miR-100 bound to m6A were significantly increased in METTL 14-overexpressing cells.Furthermore,miR-100-3p inhibitor can block the photoprotection caused by overexpression of METTL14.4.The MiRDB,miRWalK,RNA22,TargetMiner,and RNAInter databases were used to predict 38 mRNAs that may bind to miR-100-3p,which were then intersected with the mRNAs increased by UVB in the GSE119009 dataset to provide 9 mRNAs.In photoaged cells,ERRFI1 mRNA and protein levels increased.MiR-100-3p mimic can suppress ERRFI1 protein expression,and miR-100-3p inhibitor can antagonize the inhibitory effect of miR100-3p on ERRFI1 protein.A dual luciferase reporter gene assay revealed that miR-100-3p mimic could bind to the 3’-UTR region of ERRFI1 mRNA,resulting in a reduction in fluorescence intensity,whereas mutation of the region did not.5.Overexpression of METTL 14 can decrease ERRFI1 protein expression,which can be reversed by miR-100-3p inhibitor.Overexpression of ERRFI1 can inhibit the photoprotective effect of METTL 14 overexpression.ConclusionsIn this study,single UVB(30mJ/cm2)was utilized to induce photoaging in human skin fibroblasts,and it was discovered that the m6A modification level and the expression of the methyltransferase METTL14 were down-regulated in photoaged cells,while METTL14 overexpression had a photoprotective effect.METTL14 modulates the m6A level of pri-miR100,which affects DGCR8 binding and hence the subsequent cleavage and processing of primiR-100 into mature miR-100-3p.Furthermore,miR-100-3p can bind to the 3’-UTR and suppress protein expression.On the basis of METTL14 overexpression,simultaneously suppressing miR-100-3p or overexpressing ERRFI1 can inhibit the photoprotective effect of METTL14 overexpression.In conclusion,in UVB-induced photoaging of human skin fibroblasts,METTL 14-dependent m6A methylation can regulate miR-100-3p maturation via DGCR8,and skin fibroblasts photoaging through miR-100-3p-ERRFI1 axis.