ELAVL4 Encoded RNA Binding Protein HuD Controls Skin Cellular Senescence Through CDKN1A

Background: Cellular Senescence is described as a stable proliferation arrest due to telomere shorten or external senescence related stimulations during cell proliferation,and accompanied by Senescence-Associated Secretory Phenotype(SASP).The essence of skin aging is the senescence of skin cells.However,the lack of specific markers to regulate the senescence of skin cells has severely limited the anti-aging progress of skin tissue.Therefore,it is particularly important to identify new and effective markers to indicate the senescence of skin cells and to study the internal mechanism of their regulation towards skin aging.HuD(encoded by Embryonic Lethal,Abnormal Vision,Drosophila Like 4,ELAVL4)is a RNA binding protein that has been shown to be a key regulator of gene expression in neuronal system.However,there is increasing evidences that HuD plays a key regulatory roles in non-neuronal systems as well.CDKN1A(Cyclin-Dependent Kinase inhibitor-1A)is a member of the cyclin-dependent kinase inhibitor CIP/KIP family.It has been reported that HuD can bind to CDKN1 A genome thus to regulate its expression at the transcriptional level.Objective: To clarify whether ELAVL4 is involved in the regulation of skin cell senescence,and whether ELAVL4 is involved in the regulation of skin cell senescence at the transcriptional level through binding to CDKN1 A.The purpose of this study is to investigate:(1)the correlation between ELAVL4 and skin cell senescence;(2)whether ELAVL4 can regulate therapy-induced skin cell senescence;(3)whether ELAVL4 regulates skin cell senescence via CDKN1 A.Finally,based on these results,ensure whether ELAVL4 can be served as a novel marker of skin cell senescence.Methods: Blood samples(n=12)and skin tissues(n=4)were collected from clinical ex aminations of young(mean age 20)and old(mean age 75),RNA from peripheral blood mononuclear cells(PBMCs)and skin tissues were extracted by trizol method,RT-qPCR was used to detect the senescence index and the expression of ELAVL4 and their correl ation was analyzed.Different cell lines were cultured to construct Threapy-Induced Sen escence(TIS)model using bleomycin and doxorubicin.The expression level of ELAVL4 in senescent cells was detected by RT-qPCR and the expression of senescence-related genes was analyzed.In addition,SEEK co-expression database was searched and the co rrelation between ELAVL4 and SASP related gene expression was analyzed.The dermal cell senescence model induced by doxorubicin was constructed again,and ELAVL4 kno ckdown was performed using si RNA strategy.Firstly,RT-qPCR was used to clarify the knockdown of ELAVL4 and the expression of senescence related indicators and SASP fa ctors were detected.Then,we adopted the technique of immunofluorescence(IF)stainin g,SA-β-Gal(Senescence associated β-galactosidase)staining and crystal violet staining to determine whether knocking down of ELAVL4 can reverse doxorubicin induced DNA damage and skin cell senescence or not.Moreover,folic acid was used to treat cells and Western Blot was used to determine HuD expression.Furthermore,folic acid was used to treat skin cells together with CDKN1 A knockdown,and RT-qPCR,SA-β-Gal staining and crystal violet staining were used to determine whether HuD could induce CDKN1A-dependent skin cell senescence or not.Results: ELAVL4 expression was significantly higher in blood and skin in the older gro up than in the younger group(p<0.05),and the expression of ELAVL4 was positively co rrelated with the expression of CDKN1A(p<0.01).In the TIS model,the expression leve l of ELAVL4 was significantly increased(p<0.05);SEEK co-expression database showe d that the expression of ELAVL4 was significantly positively correlated with SASP facto rs CCR2,CCR9 and CCL4 in skin cell lines(p<0.05).Doxorubicin can induce senescen ce,SASP secretion and DNA damage in skin cells,but knockdown of ELAVL4 can signi ficantly reverse the senescence induced by doxorubicin.Folic acid can promote HuD ex pression.In addition,folic acid can induce senescence and SASP secretion of skin cells,and knockdown of CDKN1 A can partially reverse the folic acid-promoted senescence.Conclusion: ELAVL4 expression was significantly increased in blood,skin and senesce nt cells in the elderly.Knockdown of ELAVL4 delayed the senescence of skin cells indu ced by doxorubicin.Stabilization of HuD by folic acid could induce a CDKN1A-depend ent skin cell senescence.Therefore,our study identified ELAVL4 is a novel regulatory f actor of skin cell senescence.