The Roles And Mechanisms Of WTAP-mediated M6A Modification In Cellular Senescence Of Human Dermal Fibroblasts

Background:Cellular senescence is a state of permanent cell cycle arrest that promotes deregulated metabolism,chromatin remodeling,macromolecular damage and altered gene expression.With the increase of age,increasing senescent cells lead to the whole-body aging and the occurrence of age-related diseases.The occurrence of cellular senescence is not only regulated by genetic genes,but also by epigenetics regulation.In recent years,due to the technical progress in mass spectrometry and sequencing,RNA m6 A modification has attracted extensive attention.Studies have confirmed that m6 A modification is involved in multiple physiological processes such as RNA transport,splicing,nuclear export,stability,and translation,and plays an important role in embryonic development,tumorigenesis,and immune responses.However,the roles and mechanisms of m6 A modification in cellular senescence remains unclear.Objective:To explore the specific roles and molecular mechanisms of RNA m6 A modification and its regulators in cellular senescence of human dermal fibroblasts(HDFs).Methods:(1)Data sets from GTEx and GEO public databases were analyzed to comprehensively reveal the expression levels of m6 A regulators in skin tissues and human dermal fibroblasts of aging individuals which was confirmed by immunohistochemistry;(2)The cellular senescence models were constrcuted by subculture,UVA,H2O2,HRAS oncogene,which were used to explore the expression of WTAP and m6 A modification abundance by RT-qPCR,Western Blotting,dot blot and colorimetry;(3)The HDFs cells of stably knockdown or overexpressed WTAP gene were constructed using Lentiviral Vectors.RT-qPCR,Western Blotting,immunocytofluorescence were used to identify the effects and differences between WTAP knockdown or overexpression on the proliferation,cell cycle,SA-β-Gal staining,senescence-associated secretory phenotype and m6 A modification abundance;(4)The mRNA of downstream target was preliminary screened by RNA-seq.The target ELF3(E74 like ETS transcription factor 3),binding with WTAP was verified by MeRIP-seq,RT-qPCR,and Western Blotting.MeRIP-RT-qPCR and m6 A site prediction combined with dual-luciferase assay were used to prove WTAP mediated ELF3 mRNA m6 A modification;(5)The cellular senescence models were used to explore the expression of ELF3 by RT-qPCR;RT-qPCR,Western Blotting,immunocytofluorescence were used to identify the effects and differences between WTAP knockdown and ELF3 overexpression on the proliferation,cell cycle,SA-β-Gal staining,and senescence-associated secretory phenotype;(6)The target gene IRF8 regulated by ELF3 was screened by GO analysis and RT-qPCR,and the possible binding sites of ELF3 on the IRF8 promoter were predicted by bioinformatics methods;The binding site in IRF8 promoter was verified by Chip-qPCR;(7)RT-qPCR,Western Blotting,immunocytofluorescence were used to identify the effects and differences between IRF8 overexpression or knockdown on the proliferation,SA-β-Gal staining and senescence-associated secretory phenotype.Results:(1)Bioinformatics methods revealed that WTAP is highly expressed in aging individual skin tissue and dermal fibroblasts,which is the most closely related m6 A regulator to cellular senescence;(2)The mRNA and protein expression levels of WTAP and m6 A content were significantly increased in cellular senescence models;(3)The defect of WTAP significantly delayed the senescence of HDFs cells,while overexpression of WTAP significantly induced the senescence phenotypes of HDFs cells;(4)RNA-seq and MeRIP-Seq showed that ELF3 was the target gene of WTAP,knockdown or overexpression of WTAP can down-regulate or up-regulate ELF3 mRNA and protein levels and m6 A modification abundance;(5)The expression of ELF3 was increased in a variety of HDFs cellular senescence models;The results of RT-qPCR,Western Blotting and immunocytofluorescence showed that ELF3 induced the senescence phenotypes of HDFs;At the same time,overexpression of ELF3 aggravated the suppression of cellular senescence phenotype caused by WTAP knockdown;(6)The transcription factor ELF3 directly binded to the promoter of IRF8 and upregulated its expression level by promoting the transcriptional activity of IRF8;(7)IRF8 promoted cellular senescence phenotype;The rescue results indicated downregulating IRF8 significantly inhibited the expression of SASP caused by ELF3.Conclusions:In summary,our findings revealed that the elevated WTAP in human skin aging tissue and senescent dermal fibroblasts,overexpressed WTAP induced cellular senescence.ELF3 was involved in WTAP-mediated cellular senescence in a m6A-dependent manner.WTAP/ELF3 induced the cellular senescence through the aberrant production of SASP via upregulating IRF8.Altogether,our results determined WTAP as a potential therapeutic target for cellular senescence.