| Backgroud: Ultraviolet(UV)light is electromagnetic radiation,and is subdivided into UV-A,UV-B,or UV-C,depending on the wavelength(315–380,280–315,190–280 nm,respectively).Among these,it is accepted that excess UVB irradiation is a major risk factor for skin cancers [1].Although UVB absorption can damage various skin cell components,carcinogenesis is primarily due to photolesions formed in DNA,which are predominantly cyclobutane pyrimidine dimers(CPDs).The UV-damaged cell responds to these alterations by either inducing DNA repair activity or by inducing apoptotic death if the damage is too severe;failure of a severely damaged cell to undergo cell death can lead to the formation of an initiated tumor cell.Although,there are a lot of researchs on skin cancer induced by UVB,but the studies about the photocarcinogenesis and photodamage from the aspect of miRNA are still lacking.Mi RNAs are small endogenous noncoding RNAs that act as negative regulators of protein-coding gene expression through the binding of complementary sequences in the 3′ untranslated region(UTR)of target m RNAs.Recent studies have shown that miRNA-mediated gene silencing is mediated by a specialized family of RNA-binding proteins named Argonaute.Argonaute proteins bind these small regulatory RNAs and use their encoded sequence information to locate and silence complementary target RNAs.This binding subsequently leads to the degradation of the target RNAs and inhibition of protein synthesis.Normal miRNA expression is deregulated during skin morphogenesis as well as many types of human skin diseases,including skin cancer.Several studies have reported an association between UV radiation exposure and changes in miRNA expression in mammalian cells.In the mouse fibroblast cell line NIH3T3,UVB irradiation resulted in robust differences in miRNA expression,particularly 4 h after irradiation,suggesting that miRNAs play a role in coordinating the response to UVB stress.Similarly,UVC irradiation caused extensive changes in miRNA expression(generally increasing their abundance)in He La cells and fibroblasts,with the effects most pronounced 4 h after irradiation.Although the expression of some common miRNAs was influenced by both UVB and UVC stress(e.g.,miR-21 and miR-24),many others responded exclusively to only one or the other.However,the specific miRNAs involved in the UV-induced stress response still remain to be identified.To our knowledge,no studies of miRNA array based miRNA expression in normal human keratinocytes exposure to UVB radiation have been reported to date.In this study,we reported the results of our analysis of miRNA expression in keratinocytes subjected to UVB irradiation with dose and time-dependent manner.Recently,our research group assessed the effects of UVB irradiation on miRNA expression in keratinocytes.The miR-23a~27a~24–2 cluster,which has been reported to play a role in anti-tumorigenic pathways,DNA repair,and apoptosis,was found to be up-regulated 4 and 24 hours post-irradiation.Additionally,in another previous study by our group,we observed that miR-23 a was upregulated by a DNA repair agent(baicalin)in mouse skin irradiated with UVB light.Whether the UVB-regulated miRNAs,including miR-23 a,can influence the response to UVB-induced cell damage,such as through DNA repair,apoptosis,and growth arrest,is explored in the present study.The results of this investigation provide a model to understand the molecular regulation of miRNAs in UVB-induced cellular damage.Methods: 1.Firstly,Cultured HaCaT cells were divided into two groups,receiving either no treatment or exposure to 30 m J/cm2 of UVB,and were observed 30 min,4 h,or 24 hr later.Cell apoptosis was measured by flow cytometry,and representative flow cytometry results and quantitative analysis of cell apoptosis are shown.2.Normal human keratinocytes exposed to 30 or 60 m J ? cm2 of UVB irradiation were collected 4 or 24 h postirradiation.Global miRNA expression profiles were studied using an Agilent human miRNA microarrays,which contained probes for1223 miRNAs catalogued in the Sanger Cambridge database v10.1(http://microrna.sanger.ac.uk).We then further confirm the microarray results by real-time quantitative PCR(RT-q PCR)using miRNA sequence-specific primers.Agglomerative hierarchicaclustering analysis was performed using Cluster software and Tree View software(http://genome-www5.stanford.edu/resources/restech.shtml).3.The expression levels of mature miR-23 a,miR-27 a and miR-24 4 h and 24 h after 30 m J/cm2 UVB irradiation(detected by Q-RT-PCR).4.Prior to irradiation with 30 m J/cm2 UVB,cultured HaCaT cells were treated with miR-23 a mimic(50 n M),miR-23 a mimic control(50 n M),miR-23 a inhibitor(50 n M),or miR-23 a inhibitor control(50 n M).One UVB-exposed group did not undergo treatment The levels of UVB-induced CPDs in cells 30 min and 24 h post-irradiation were measured by immunofluorescence as well as by southwestern dot-blots with an anti-CPD antibody.Representative CPD immunofluorescence images and quantitative analysis of CPD levels are shown.5.Cultured HaCaT cells were pre-treated with vehicle,miR-23 a mimic(50 n M),miR-23 a mimic control(50 n M),miR-23 a inhibitor(50 n M),or miR-23 a inhibitor control(50 n M),and then underwent 30 m J/cm2 UVB irradiation.One UVB-exposed group did not undergo treatment,and an additional control group was not exposed to UVB and did not undergo treatment.6.Mutations in two predicted miR-23 a binding sites in TOP1 3’ UTR.The cells were plated in six wells and incubated until 70% confluent and transfected with one of the following 12 combinations:(1)TOP1 3’ UTR(638-645);(2)TOP1 3’ UTR(1020-1027);(3)TOP1 3’ UTR with miR-23 a 638-645 seed-matching mutation(TOP1 3’ UTR(638-645)mt);(4)TOP1 3′UTR with miR-23 a 1020-1027 seed-matching mutation(TOP1 3’ UTR(1020-1027)mt);(5)TOP1 3’ UTR(638-645)+ sc-miR(miRNA scramble control);(6)TOP1 3’ UTR(1020-1027)+ sc-miR;(7)TOP1 3’ UTR(638-645)mt + sc-miR;(8)TOP1 3’ UTR(1020-1027)mt + sc-miR;(9)TOP1 3’ UTR(638-645)+ miR-23a;(10)TOP1 3’ UTR(1020-1027)+ miR-23a;(11)TOP1 3’ UTR(638-645)mt + miR-23a;and(12)TOP1 3’ UTR(1020-1027)mt + miR-23 a.Both firefly luciferase and Renilla luciferase activities were determined in the HaCaT cell line.Firefly luciferase activity was then normalized with Renilla luciferase activity in the same well.7.Mi R-23 a mimic decreased the protein expression of TOP1,Caspase-7 and STK4 protein in HaCaT cells when compared to UVB or mimic-control groups,while miR-23 a inhibitor increased protein expression of TOP1,Caspase-7 and STK4 in HaCaT cells relative to UVB or inhibitor-control groups.Quantitative analysis of TOP1,Caspase-7 and STK4 protein levels are shown.Results: 1.Flow cytometry and cell immunofluorescence were used to detect cells at 30 min,4 h,and 24 h after exposure to 30 m J/cm2 UVB radiation.The results showed an increase in apoptotic cells after UVB irradiation,with the number of apoptotic cells at 24 h greater than that at 4 h.The apoptotic rates of the different groups are listed.Increased levels of CPDs were observed at 30 min and 24 h post-irradiation,and the abundance of CPDs in cells at 24 h post-irradiation is significantly decreased when compared with the levels at 30 min post-irradiation.2.And then miRNA expression array was performed to study the expression of 1223 human mature miRNAs.The analysis revealed a significant number of miRNAs that were dysregulated after radiation exposure in comparison to non-irradiated keratinocytes.Totally 45 miRNAs were upregulated or downregulated more than two-fold either after 30 or 60 m J/cm2 of UVB irradiation at each time point.Among them,30 miRNAs were further confirmed by real-time RT-PCR.Next,the unsupervised hierarchical clustering analysis of miRNA expression was performed.The analysis grouped samples by time of collection after radiation(4 hours vs.24 hours),but not by irradiation dose:(i)miRNAs downregulated at 4 hours and returned tonormal or to upregulated level at 24 hours:hsa-miR-326,hsa-miR-423-5p,hsa-miR-193 b,and hsa-miR-542-5p;(ⅱ)miRNAs upregulatedat 4 hours and returned to normal or downregulated level at 24 hours :hsa-miR-26 a,hsa-let-7c,hsa-let-7f,hsa-miR-487 b,hsa-miR-26a-2,hsa-miR-543,and hsa-miR-487b;(ⅲ)miRNAs upregulated at both time points:(hsa-miR-31,hsa-miR-24,hsa-miR-27 b,hsa-let-7b,hsa-miR-200 b,hsa-miR-125 b,hsa-miR-27 a,hsa-let-7g,hsa-miR-23a;hsa-miR-98,hsa-miR-221,hsa-miR-186,hsa-miR-30 a,hsa-miR-22,hsa-miR-96,hsa-miR-16,hsa-miR-18 b,hsa-miR-34 a,hsa-let-7a,hsa-miR-93,hsa-miR-185,hsa-miR-197,hsa-miR-365,hsa-miR-23 b,hsa-miR-29a;(iv)miRNAs downregulated at both time points:hsa-miR-489,hsa-miR-138-1,hsa-miR-138-2,hsa-miR-23 a,hsa-miR-296-5p,hsa-miR-376 b,hsa-miR-493,hsa-miR-126,hsa-miR-143.3.We found that the expression levels of miR-23 a and miR-27 a in HaCaT cells increased 4 h after exposure to 30 m J/cm2 UVB,and returned to endogenous levels 24 h following irradiation.The expression of miR-24 was consistently up-regulated in HaCaT cells both 4 h and 24 h after UVB irradiation.4.Twenty-four hours after 30 m J/cm2 UVB irradiation,cell viability was reduced significantly.In cells exposed to UVB irradiation,over-expression of miR-23 a had a protective effect on cell viability,while inhibition of miR-23 a after UVB irradiation resulted in a slight decrease in viability compared to other groups.However,the same effect was not found in the miR-27 a and miR-24 intervention groups.5.UVB exposure resulted in a marked increase in CPD immunofluorescence in HaCaT cells at 30 min and 24 h post-irradiation.Cells with over-expression of miR-23 a prior to UVB irradiation showed an obvious decrease in CPD levels 24 h post-irradiation,while inhibition of miR-23 a resulted in a relatively higher number of CPD-positive cells.Statistical analysis also found that the efficiency of CPD removal was greater in the group treated with miR-23 a mimics than in the UVB control group.6.We used Hoechst staining/flow cytometry as well as Caspase-3 activity(an early marker of apoptosis)to determine the effect of miR-23 a on HaCaT cell apoptosis.The results show that miR-23 a mimics significantly decreased UVB-induced cellapoptosis(P < 0.01).In contrast,cell apoptosis was increased after treatment with a miR-23 a inhibitor(P < 0.0001).In addition,control oligos(scrambled oligos)had no observable effects on cell apoptosis(P < 0.05).The apoptotic rates of the different groups are listed.The results show an increase in the number of apoptotic cells after UVB irradiation.Caspase-3 activity was strongly induced in HaCaT cells following exposure to 30 m J/cm2 UVB,but decreased in cells transfected with miR-23 a.As shown,the miR-23 a mimic significantly reduced caspase-3 activity that was induced by UVB;transfection with a miR-23 a inhibitor reversed this effect.These results clearly indicate that miR-23 a plays an important role in preventing the induction of apoptosis in UVB-exposed HaCaT cells.7.The TOP1 3’ UTR was fused with a luciferase reporter and mutations in two predicted miR-23a-binding sites were introduced in the 3’ UTR.According to analysis performed with a dual-luciferase assay system,the luciferase activity of the construct containing a mutation in the TOP1 3’ UTR at one predicted binding site(638-645)was decreased after miR-23 a transfection,while the other TOP1 3’ UTR binding mutant(1020-1027)did not demonstrate a significant influence on the luciferase activity.This suggests that miR-23 a can decrease luciferase activity of TOP1 3’ UTR by sequence-specific base pairing with the 638-645 3’ UTR of TOP1.Both gain-of-function and loss-of-function approaches were employed to further verify that TOP1 is a target gene of miR-23 a in HaCaT cells.Over-expression of miR-23 a significantly decreased m RNA expression of TOP1.In contrast,inhibition of miR-23 a expression increased TOP1 m RNA levels.Regulation of the expression of TOP1 by miR-23 a mimics and a miR-23 a inhibitor was further confirmed at the protein level by Western blotting.Taken together,these results strongly suggest that,in HaCaT cells,TOP1 is a target gene of miR-23 a.8.In silico analysis revealed that Caspase-7 and STK4,two critical mediators of apoptosis signaling,are potential targets of miR-23 a.Accordingly,the 3’ UTRs of Caspase-7 and STK4 m RNA were found to contain predicted binding sites formiR-23 a.To confirm this,we transfected UVB-irradiated cells(30 m J/cm2)with a miR-23 a mimic,a mimic control,a miR-23 a inhibitor or an inhibitor control.As a control,some cells were either transfected with vehicle or were not exposed to UVB radiation.We then determined the m RNA and protein levels of Caspase-7 and STK4 by q RT-PCR and Western blotting,respectively.As shown,UVB irradiation increased Caspase-7 and STK4 expression.Both gain-of-function and loss-of-function approaches were employed to further verify that Caspase-7 and STK4 were target genes of miR-23 a in HaCaT cells.Over-expression of miR-23 a significantly decreased m RNA expression of both Caspase-7 and STK4.In contrast,inhibition of miR-23 a expression increased the m RNA levels of both.Regulation of the expression of Caspase-7 and STK4 by miR-23 a mimics and a miR-23 a inhibitor were further confirmed at the protein level by Western blotting.Taken together,these results strongly suggest that,at least in HaCaT cells,Caspase-7 and STK4 are both target genes of miR-23 a.Conclusion: In summary,We observed several specific patterns of miRNA response to UVB irradiationin HaCaT cells,miR-23 a is sensitive to UVB irradiation.Mi R-23 a protects against UVB-induced injury to HaCaT cells via its regulation of potential target genes,Caspase-7,STK4,and TOP1.These novel findings may have extensive implications for the diagnosis and treatment of a variety of conditions caused by UVB-induced photodamage,such as sunburns,photoaging,and cancer. |
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