Effect And Mechanism Of Mettl14 And Mettl3 On Epidermal Stem Gells

Objective:The balance between self-renewal and differentiation of epidermal stem cells are regulated by various genes.The RNA metabolism process regulates the genes' expression.RNA metabolism,including the mRNA stability,splicing and translational efficiency,is regulated by Mettl14 and Mettl3.As the key components of the methyltransferase complex,the Mettl14 and Mettl3 mediated m6A modification regulates the RNA metabolism,while their effects on epidermis remain unknow.In this study,we investigate the functions of Mettl14 and Mettl3 on the self-renewal and differentiation of epidermal stem cells and the effect of Mettl3 on epidermal cancerMethods:1.We use conditional gene knockout technology to specifically silence the Mettl14 in epidermis and investigate the phenotype of mutant mice using various immunohistochemistry and skin transplantation.2.The primary keratinocytes isolated from Mettl14 mutant mice were detected for m6A level.The RNA-seq and qPCR were used for screening and validating the potential downstream targets 3.We generated the Mettl3 specific deletion mice,and investigate the phenotype using immunohistochemistry.4.We construct the shRNA-mediated knockdown Mettl3 in A431,a cutaneous squamous cell carcinoma(cSCC)cell line.And we investigate the the phenotype of cancer cells and the expression of genes related to self-renewal and differentiation.We also study the effect of knockdown Mettl3 using in vivo xenograft tumor modelsResults:1.The epidermis of Mettl14 mutant mice are thicker on the postnatalday 7,the proliferation of epidermal basal cells is increased.The mutant mice died about postnatal day 7-9 Following the skin transplantation onto nude mice,the epidermis phenotype on post-operation day 14 is similar to the mutant mice on postnatal day 7.The epidermal proliferation decreases on the post-operation day 17,and the epidermis are total lost on the post operation day 34.2.The m6A level of primary keratinocytes isolated from Mettl14 mutant mice is decreased and the upregulated expressions of S100a8 and S100a9 are verified by and qPCR.3.The epidermis of Mettl3 mutant mice are also thicker and the basal cells are hyperproliferative on the postnatal day 9.The expression of Mettl14 is decreased in the Mettl3 mutant mice.4.The expression of Mettl14 and the m6A level are also decreased in the Mettl3 knockdown A431 cells.The Mettl3 knockdown protomes the cSCC cell differenation,inhibits the stemness of cSCC cells.The Mettl3 also inhibits the tumor growth in vivoConclusion:1.Mettl14 and Mettl3 are the key component of the m6A methyltransferase complex Conditional knockout the Mettl14 in epidermal keratinocytes can lead to transient hyperproliferation of epidermal stem cells and eventually lead to depletion of stem cell pool.2 The Mettl3 knockdown in A431 cells,a human cutaneous squamous cell carcinoma cell line,can inhibit the tumor growth.3.Our study preliminarily elucidates the effects and mechanisms of Mettl14 and Mettl3 on the self-renewal and differentiation of epidermal stem cells and cutaneous squamous cell carcinoma.These contribute to the understanding of epidermal homeostasis-related diseases(trauma,epidermal cancer)...