Functional And Mechanistic Study Of Long Noncoding RNA In Ultraviolet Induced Skin Photodamage

Skin damage caused by ultraviolet(UV)radiation can be divided into two groups:acute damage which is mainly characterized by skin inflamation,DNA damage and immune supression,and chronic damage which is mainly characterized by photoaging and skin carcinogenesis.With the destruction of the ozone layer,the increase in UVB radiation on earth is a major environmental threat to the skin,increasing the risk of damage with long-term consequences.Protection from UV radiation has become an important aspect of people's daily life.Recent evidences have shown that lncRNAs,which are defined as RNAs longer than 200 nucleotides without protein-coding potential,play critical roles in many important biological processes.Mutations and dysregulations of these lncRNAs would contribute to the development of various complex diseases.However,research on the effects of UV radiation on lncRNA expression in HDF is limited.The aim of this study was to identify changes in the expression profile of lncRNAs in HDF after UVB radiation and uncover the function and mechanism of lncRNAs in ultraviolet induced skin damage,which may provide a new target of prevention and treatment of skin photodamage disease.Firstly,we adopted high-throughput RNA sequencing to compare the differences both in mRNA and lncRNA expression profiles in UVB irradiated HDF with non-irradiated HDF.Using a filtering criteria of P ? 0.05,we found 61 lncRNA transcripts(56 up-regulated and five down-regulated)and 862 mRNA transcripts(577 up-regulated and 285 down-regulated)were significantly altered in HDF with UVB radiation,compared with the control groups of HDF.Then we performed comprehensive bioinformatics analyses including gene ontology(GO)term enrichment analyses and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analyses to explore the roles of differentially expressed lncRNAs.We explored the potential functions of dysregulated UVB-responsive lncRNAs by targeting either cis-or trans-regulated target protein-coding genes.By KEGG enrichment analysis of lncRNAs and mRNAs,we identified related pathways including TNF signaling pathway,P53 signaling pathway,and DNA damage repair.The high consistency between the predicted functions of dysregulated lncRNAs and the functions of dysregulated mRNAs indicated that lncRNAs play critical roles in regulating the expression of protein-coding genes.Finally,we randomly selected several differently expressed lncRNA transcripts and analyzed their expression levels by qRT-PCR to confirm the reliability of the RNA sequencing data.The qRT-PCR results were consistent with the RNA-Seq data,with the same trends observed for each lncRNA,confirming the accuracy and reliability of the RNA sequencing analysesIn conclusion,this study represents the first analysis to compare the differences in lncRNA expression profiles in UVB irradiated HDF with non-irradiated HDF,which have identified some lncRNAs associated with UVB induced skin photodamage.These findings lay a foundation for future investigations into the expression patterns of lncRNAs with roles in the response to UV radiation and in skin acute photodamage,which may provide new insights for the development of novel preventive and therapeutic approaches against skin photodamage.The aging of human skin is caused by genetic and environmental factors.Among environmental factors,solar ultraviolet radiation are the main factors,causing atrophy of the skin,coarse wrinkles and leathery skin.LncRNAs are involved in various biological processes,however,their potential implications in skin photoaging remain virtually unexplored.In the previous study,we performed high-throughput RNA sequencing to characterize the lncRNA profiles in UVB-irradiated primary human dermal fibroblasts(HDF),and found 61 lncRNAs were significantly altered.In this study,we choose lncRNA RP11-670E13.6,one of the differently expressed lncRNAs in UVB-irradiated HDF,to further investigated its function and regulatory mechanism in ultraviolet induced skin photoaging.Firstly,we performed qRT-PCR analysis and found that lncRNA RP11-670E13.6 was significantly elevated at different time after UVB irradiation.Then we performed measure telomere length,?-galactosidase staining,cell viability analysis,cell cycle analysis to explore the roles of lncRNA RP11-670E13.6 in cellular senescence.The results showed that knocking down of lncRNA RP11-670E13.6 promoted a robust senescence phenotype,including mean telomere length and cell proliferation decreased,numbers of senescence-associated ?-galactosidase-positive cells increased,accumulation of cells in G0/G1 phase.We also detected intracellular ROS,catalase(CAT)and superoxide dismutase(SOD)activity,analyzed apoptosis in lncRNA RP11-670E13.6 depleted HDF,which showed no significantly differences.Moreover,immunofluorescence and western blot analysis showed that lncRNA RP11-670E13.6 may facilitate DNA damage repair by increasing ATM and yH2A.X protein levels.In addition,western blot analysis showed that knocking down of lncRNA RP11-670E13.6 activated the p16-pRB pathway.Therefore,we propose that lncRNA RP11-670E13.6 may delay cellular senescence in UVB damaged HDF through the p16-pRB pathway.Next,we further study the underlying mechanisms through which lncRNA RP11-670E13.6 regulated cellular senescence.Using cytoplasmic and nuclear RNA fractions from HDF,we observed that lncRNA RP11-670E13.6 translocated from the nucleus to the cytoplasm after UVB irradiation,which confirmed the results of fluorescence in situ hybridization.By bioinformatics statistical analysis,we identified miR-663a having putative binding sites with IncRNA RP11-670E13.6,CDK4 and CDK6.Besides,we also found that miR-663a inhibited the proliferation and stimulated apoptosis of HDF.Cell cycle analysis showed that miR-663a inhibitor drove progression beyond the G1/S transition in UVB-irradiated HDF.Dual-luciferase assays,western blot and qRT-PCR showed that miR-663a directly bound to the 3'-UTR of CDK4 and CDK6 mRNA and inhibited cdk4 and cdk6 protein expression.Moreover,lncRNA RP11-670E13.6 can bound to miR-663a and that the binding sites were vital for reciprocal repression of lncRNA RP11-670E13.6 and miR-663a.In addition,using RNA pull-down assays,RNA immunoprecipitation assays,western blot analysis,we found that UVB irradiation down-regulated hnRNPH expression,which directly bound to and suppressed lncRNA RP11-670E13.6 expression.Collectively,this study revealed for the first time the specific function and mechanism of lncRNA RP11-670E13.6 in the process of UVB-induced skin photodamage.Knocking down of lncRNA RP11-670E13.6 promoted cellular senescence.Briefly,hnRNPH physically interacted with IncRNA RP11-670E13.6 and blocked its expression under physiological conditions.When UVB irradiation down-regulated hnRNPH,lncRNA RP11-670E13.6 expression was significantly increased in a ROS-independent manner and facilitating DNA damage repair by increasing the kinase activity of ATM and the phosphorylation of histone H2A.X molecules.Moreover,upon UVB irradiation,lncRNA RP11-670E13.6 translocated from the nucleus to the cytoplasm.In the cytoplasm,lncRNA RP11-670E13.6 functioned as an endogenous "sponge" by directly binding to miR-663a,abolishing the repressive activities of miR-663a on Cdk4 and Cdk6,and thereby delaying UVB-induced cellular senescence.