| Objectives:Skin is an ideal organ to observe and analyze the internal and external driving factors of aging.A number of studies have explored various cellular signaling pathways that influence the fate of epidermal cells,but little is known about how the biological dynamics of human epidermal keratinocytes change from fetus to birth to aging.This study aimed to compare the biological characteristics of skin tissues and keratinocytes at three different developmental stages including fetal,childhood and aged stage.RNA-Seq and bioinformatics analysis were conducted to study the molecular characteristics of keratinocytes during development and aging process,and potential key genes related to aging were also screened.Methods:(1)Fetal,childhood and aged skin tissues were collected.Epidermal thickness was compared by HE staining and expression of keratin KRT14 and KRT10 was compared by immunofluorescence staining.Skin senescence was also evaluated by SA-β-Gal staining.(2)Morphological and SA-β-Gal staining were performed on fetal,childhood and aged human keratinocytes.The expression of keratin differentiation genes KRT1 and KRT10,and those of published skin aging-related genes reported were compared by q RT-PCR.(3)RNA-Seq was used to compare the molecular characteristics of human fetal,childhood and aged skin keratinocytes.Key genes closely related to skin development,differentiation and aging were screened using GO database combined with the published single-cell sequencing results.Results:(1)Through the comparison of fetal,childhood and aged skin tissues,it was found that the epidermis become gradually thickened and the expression of KRT10,keratinocyte differentiation marker,was gradually enhanced with the increase of age.In addition,there were more SA-β-Gal positive senescent cells in the aged skin.(2)Primary keratinocytes in fetal,childhood and aged stages were isolated and cultured in vitro.There were more flat and enlarged keratinocytes in the aged groups while the keratinocytes in fetal and childhood groups were more uniform,closely packed and clearly defined.Compared with the fetal and childhood groups,there were a higher proportion of SA-β-Gal positive senescent keratinocytes in the aged group,expressing relatively higher levels of keratin differentiation genes KRT1 and KRT10,and lower levels of age-related down-regulated genes HES1,FOS and KLF6,which was consistent with the results of previous single cell RNA-Seq studies.(3)Through high-throughput RNA-Seq and bioinformatics analysis,we found that keratinocytes in childhood and aged groups were more similar compared to the fetal groups.The differential genes screened by pairings of three groups of samples were enriched in 4 items:epidermal development,skin development,keratinocyte differentiation and keratinization.The three groups of samples showed distinct patterns in the expression of the genes involved in these items and the keratin family genes,suggesting that the key genes involved in these biological processes show dynamic changes in keratinocytes at different developmental stages.In addition,the single cell RNA-Seq results of published studies were also verified in the RNA-Seq of this study,indicating that HSPA1 B,DNAJB1 and PHLDA2 genes might be involved in the development and senescence of keratinocytes.The transcription factors EHF,MAFB,KLF4,SNAI2,TP53,KDM1 A and LEF1 in the three groups of samples also showed a downward trend with the increase of age,suggesting that they may be the key factors involved in regulating epidermal development and aging.Conclusions:The skin tissue and keratinocytes in the fetal,childhood and elderly groups showed quite different biological properties and patterns of dynamic changes.The transcriptomics of keratinocytes were more similar in the childhood and the elderly groups compared to the fetal ones.Moreover,some aging-related genes screened by previous single-cell sequencing study were also validated.This study can lay a foundation for further exploration of the specific regulatory mechanisms of epidermal development and aging,and provide references for in-depth research on age-related targets and key pathways.It can also help to develop new drugs anti-aging drugs and for the prevention and treatment of skin tumors,and can provide a reference for the anti-aging research of other tissues and organs.This study would provide reference for development of new anti-aging therapy and for prevention and treatment of age-related skin diseases. |
