【作者】 王颖；

【导师】 李永芳；

【作者基本信息】 中国医科大学 ， 公共卫生（专业学位）， 2025， 硕士

【摘要】 目的:中波紫外线（Ultraviolet B,UVB）是确认的环境致癌因子,其可穿透皮肤表面作用于角质形成细胞,引发光损伤与癌变。研究表明,UVB暴露导致的细胞凋亡是诱发皮肤损伤的重要病理机制,且这一过程伴随DNA损伤。毛细血管扩张性共济失调症突变（Ataxia-telangiectasia mutated,ATM）激酶以及ATM和Rad3相关（ATM and RAD3-related,ATR）激酶在DNA损伤修复反应中发挥主导作用。ATM/ATR活化被认为是抵御UVB所致DNA损伤的核心事件。转录因子NF-E2相关因子1（Nuclear factor-erythroid 2 related factor 1,NFE2L1,简称NRF1）属于碱性亮氨酸拉链CNC亚家族（CNC basic-leucine-zipper,CNC-b ZIP）,可在多种外源性因素暴露条件下入核活化。课题组前期研究发现NRF1基因沉默会加剧UVB诱导的人角质形成细胞（HaCaT）DNA损伤,且伴随了ATM/ATR的磷酸化水平下降。然而,转录组测序提示NRF1沉默未影响ATM/ATR的基因表达。基于DNA损伤修复反应依赖于蛋白-蛋白之间的直接结合,我们推测,NRF1可能通过蛋白互作直接参与调控ATM/ATR的活化,从而抵御UVB所致DNA损伤。在本研究中,我们以UVB所致细胞凋亡及DNA损伤为出发点,利用分子对接、免疫荧光共定位和免疫沉淀技术阐明NRF1与ATM、ATR的相互作用,并结合ATR磷酸化突变体挽救实验验证核心假说,以期得到的数据为挖掘UVB所致皮肤损伤的新靶点提供理论依据,为理解NRF1的生物学功能提供新证据。研究方法:1、细胞培养:角质形成细胞铺于37℃培养箱进行培养。细胞贴壁后,野生型细胞采用DMEM（Dulbecco’s Modified Eagle Medium）基础培养基,添加10%胎牛血清（Fetal Bovine Serum,FBS）,1%青霉素-链霉素（Penicillin-Streptomycin）进行培养;对于携带靶向NRF1 sh RNA慢病毒（NRF1 knockdown,NRF1-KD）以及非靶标阴性对照慢病毒（Scramble）的HaCaT细胞,则在野生型细胞培养基的基础上添加0.5μg/m L嘌呤霉素进行培养。当细胞生长达到80%时,进行消化传代,并收集样品。2、UVB暴露后细胞凋亡水平分析:以野生型HaCaT细胞为研究对象,给予不同剂量的UVB照射（Control、10、20、30 mJ/cm²）,利用Western Blot检测凋亡相关分子cleaved PARP的蛋白表达;利用Annexin V-FITC/PI双染结合流式细胞仪检测凋亡细胞比例。为明确NRF1沉默对上述凋亡指标的影响,以Scramble细胞和NRF1-KD细胞为研究对象,给予20 mJ/cm²的UVB照射,评估凋亡指标的变化。3、UVB暴露后NRF1、ATM及ATR蛋白表达水平分析:以野生型HaCaT细胞为研究对象,采用不同剂量的UVB（Control、10、20、30 mJ/cm²）照射,提取细胞核,利用Western Blot检测核内NRF1、ATM及ATR蛋白的表达。4、DNA损伤评估:利用免疫荧光技术检测UVB暴露导致的特异性DNA加合物环丁烷嘧啶二聚体（Cyclobutane pyrimidine dimer,CPD）的荧光强度,评估DNA损伤程度。5、共定位实验:通过免疫荧光染色观察NRF1与ATM、ATR在细胞核内的空间分布一致性,利用免疫荧光染色观察NRF1与DNA损伤相关蛋白CPD、磷酸化组蛋白（Phosphorylated Histone H2AX,γ-H2AX）在细胞核内的空间分布一致性,利用Image J软件进行定量分析。6、HADDOCK分子对接预测:本研究通过Uni Prot数据库获取NRF1、ATM及ATR蛋白的晶体结构数据,将标准化的PDB格式文件输入HADDOCK平台进行对接预测,输出前十个对接位点的结合分数。7、免疫共沉淀（Co-IP）实验:在对照和UVB处理的HaCaT细胞中,使用ATM特异性抗体下拉蛋白,利用所得到的蛋白对NRF1进行免疫印迹分析;再使用NRF1特异性抗体下拉蛋白,利用所得到的蛋白对ATM进行免疫印迹分析,以明确NRF1和ATM的蛋白互作。NRF1和ATR的蛋白互作方法同ATM。8、激活型磷酸化突变体挽救实验:运用定点突变技术,将ATR的428位丝氨酸替换为天冬氨酸（ATR-S428D）,模拟磷酸化过程。在NRF1-KD细胞中分别转染突变体(ATR^mutant)和对照质粒(ATR^vector),利用Western Blot检测p-ATR（Ser428）的蛋白水平,评估质粒构建是否成功;之后将转染突变体的细胞给予20 mJ/cm²的UVB照射,检测CPD的荧光强度,评估ATR磷酸化水平的持续活化是否能挽救NRF1沉默对DNA损伤的影响。9、统计分析:本研究所有数据均使用Graph Pad Prism 10软件进行数据分析,多组间比较采用单因素或双因素方差分析,组间两两比较采用Bonferroni法。结果:1、不同剂量UVB暴露可导致细胞凋亡:野生型HaCaT细胞暴露于不同剂量的UVB（Control、10、20、30 mJ/cm²）24小时,结果发现与Control相比,其他剂量组的Cleaved PARP的表达水平显著升高（P<0.05）,且在20 mJ/cm²变化的最为显著。Annexin V/PI双染的结果显示20 mJ/cm²的UVB处理后凋亡细胞所占的比例较高（P<0.05）,提示UVB暴露可以导致细胞凋亡,且20mJ/cm²可能是UVB诱导细胞凋亡的较佳剂量。2、不同剂量UVB暴露可导致NRF1蛋白入核:野生型HaCaT细胞暴露于不同剂量的UVB（Control、10、20、30 mJ/cm²）,通过Western Blot检测细胞核内NRF1的表达水平,结果显示与Control组相比其他剂量组的NRF1表达水平显著升高（P<0.05）,且20 mJ/cm²的UVB处理后NRF1的表达显著上调（P<0.05）。3.NRF1沉默加重UVB暴露诱导的细胞凋亡:Scramble和NRF1-KD的HaCaT细胞,分别用20 mJ/cm²的UVB处理,结果显示NRF1-KD细胞的Cleaved PARP表达显著上调（P<0.05）,表明NRF1沉默可以加重UVB所致细胞凋亡。4.NRF1与DNA损伤相关蛋白存在共定位:野生型HaCaT细胞给予UVB暴露,采用免疫荧光技术分析NRF1与CPD和γ-H2AX的空间定位。结果显示,经20mJ/cm²的UVB处理后,NRF1与γ-H2AX在细胞核内显著共定位。同时,NRF1与CPD阳性位点亦存在空间共定位,提示其可能通过靶向DNA损伤位点参与调控修复过程。5.UVB暴露可诱导核内ATM与ATR蛋白活化:通过Western Blot技术检测野生型HaCaT细胞核内ATR、p-ATR、ATM和p-ATM的表达水平,结果显示20 mJ/cm²的UVB处理后,ATR、p-ATR、ATM和p-ATM的表达显著上调（P<0.05）,表明UVB暴露可诱导细胞核内ATM和ATR的活化。6.蛋白分子对接结果表明ATM和ATR与NRF1有可结合位点:蛋白分子对接分析结果显示,NRF1与ATM和ATR均存在蛋白结合可能。7.免疫荧光提示NRF1和ATM、ATR之间存在共定位:野生型HaCaT细胞给予UVB暴露,采用免疫荧光技术分析NRF1与ATM、ATR的空间定位。结果显示,经20 mJ/cm²的UVB处理后,NRF1与ATM、ATR在细胞核内显著共定位。8.NRF1和ATM、ATR之间存在直接的蛋白互作:野生型HaCaT细胞暴露于20 mJ/cm²的UVB,免疫共沉淀实验发现,ATM下拉蛋白中能检测到NRF1,在NRF1下拉的蛋白中能检测到ATM,提示ATM和NRF1存在直接结合。ATR的免疫共沉淀结果与ATM相同,提示ATR和NRF1也存在直接结合。9.ATR的磷酸化突变体转染可以挽救NRF1-KD细胞UVB暴露所致DNA损伤:文献提示UVB暴露的直接响应蛋白是ATR,于是我们在NRF1-KD细胞中转染ATR磷酸化突变体。首先,利用Western Blot技术检测p-ATR的表达水平,结果显示ATR磷酸化突变体转染后,NRF1-KD细胞中p-ATR的表达显著上调（P<0.05）,提示转染成功。之后,给予细胞20 mJ/cm²UVB暴露,与Scramble细胞相比,NRF1-KD细胞的CPD荧光强度显著升高,但是与转染ATR^vector的NRF1-KD细胞相比,转染了ATR^mutant的NRF1-KD细胞CPD表达显著下调（P<0.05）,表明ATR的持续磷酸化可以挽救NRF1沉默所致DNA损伤加重。结论:1、NRF1入核活化可抵御UVB所致细胞凋亡。2、NRF1可与ATM及ATR直接结合,并通过调控ATR活性抵御UVB所致DNA损伤。更多还原

【Abstract】 Objective:Ultraviolet B（UVB）is a confirmed environmental carcinogen that penetrates the skin surface to act on keratinocytes,inducing photodamage and carcinogenesis.Studies have shown that UVB-induced cell apoptosis is a critical pathological mechanism underlying skin injury,and this process is accompanied by DNA damage.The ataxia-telangiectasia mutated（ATM）kinase and ATM and RAD3-related（ATR）kinase play dominant roles in the DNA damage response（DDR）.Activation of ATM/ATR is considered a core event in defending against UVB-induced DNA damage.Nuclear factor-erythroid 2 related factor 1（NFE2L1,abbreviated as NRF1）,a member of the CNC basic-leucine-zipper（CNC-b ZIP）subfamily,can translocate into the nucleus and activate under various exogenous exposure conditions.Previous studies by our group showed that NRF1 gene silencing exacerbates UVB-induced DNA damage in human keratinocytes（HaCaT）,accompanied by decreased phosphorylation levels of ATM/ATR.However,transcriptome sequencing indicated that NRF1 silencing did not affect the gene expression of ATM/ATR.Given that DDR relies on direct protein-protein interactions,we hypothesized that NRF1 might be modulated ATM/ATR activation through protein-protein interactions and then to defensed UVB-induced DNA damage.In this study,from the view of UVB-induced cell apoptosis and DNA damage,we aimed to elucidate whether NRF1 interacted with ATM/ATR by molecular docking,immunofluorescence colocalization,and co-immunoprecipitation techniques.The ATR phosphorylation mutant rescue assay was also performed to validate the core hypothesis.We expected that all data could provide a theoretical basis for identifying new targets of UVB-induced skin damage and offer new evidence for understanding the biological functions of NRF1.Methods:1.Cell Culture:HaCaT keratinocytes were cultured in a 37°C incubator.For wild-type cell line,Dulbecco’s Modified Eagle Medium（DMEM）was supplemented with 10%fetal bovine serum（FBS）and 1%penicillin-streptomycin.For cells transfected with lentivirus carrying NRF1-targeting sh RNA（NRF1-KD）or non-targeting scrambled sh RNA（Scramble）,the culture medium was supplemented with0.5μg/m L puromycin.Cells were passaged at 80–90%confluency and harvested for subsequent experiments.2.Analysis of Apoptosis Levels:Wild?type HaCaT cells were subjected to different doses of UVB irradiation（Control,10,20 and 30?mJ/cm²）.Cleaved PARP protein expression,an apoptosis?related marker,was assessed by Western blot,and the proportion of apoptotic cells was determined by Annexin V?FITC/PI double staining.To clarify the impact of NRF1 silencing on these apoptosis parameters,Scramble and NRF1?KD cells were exposed to 20?mJ/cm²UVB,and the aforementioned apoptosis indicators were evaluated.3.Analysis of Nuclear NRF1,ATM,and ATR Protein Expression:Wild?type HaCaT cells were irradiated with UVB at 0（Control）,10,20,or 30?mJ/cm².Nuclear extracts were prepared,and NRF1,ATM,and ATR protein levels were determined by Western blot.4.DNA Damage Assessment:Immunofluorescence staining was used to detect the fluorescence intensity of cyclobutane pyrimidine dimers（CPDs）,specific DNA adducts induced by UVB exposure,to evaluate the extent of DNA damage.5.Colocalization Assay:Immunofluorescence staining was performed to examine the spatial colocalization of NRF1 with ATM and ATR in the nucleus.Additionally,colocalization of NRF1 with DNA damage markers,cyclobutane pyrimidine dimers（CPDs）and phosphorylated histone H2AX（γ-H2AX）,was assessed.Quantitative analysis was conducted using Image J software.6.HADDOCK Molecular Docking Prediction:The crystal structure data of NRF1,ATM,and ATR proteins were obtained from the Uni Prot database.Standardized PDB-format files were submitted to the HADDOCK platform for docking prediction,and then the confidence score of the top 10 potential binding sites were exported.7.Co-immunoprecipitation（Co-IP）Assay:In both control and UVB-treated HaCaT cells,proteins were immunoprecipitated using an ATM-specific antibody,followed by immunoblotting for NRF1 to assess their interaction.Similarly,NRF1-specific antibody was used for immunoprecipitation,and the precipitated proteins were analyzed for ATM by immunoblotting to confirm the NRF1–ATM interaction.The same procedure was applied to investigate the interaction between NRF1 and ATR.8.Rescue Experiment Using Active Phospho-mimetic Mutant:Site-directed mutagenesis was employed to substitute serine 428 of ATR with aspartic acid（ATR-S428D）to mimic phosphorylation.NRF1-KD cells were transfected with either the mutant plasmid(ATR^mutant)or control vector(ATR^vector).Western blot was used to detect p-ATR（Ser428）levels to confirm successful plasmid construction.Transfected cells were then exposed to 20?mJ/cm²UVB,and CPD fluorescence intensity was measured to evaluate whether sustained activation of ATR phosphorylation could rescue the DNA damage phenotype caused by NRF1 silencing.9.Statistical Analysis:All data in this study were analyzed using Graph Pad Prism10 software.One-way or two-way ANOVA was used for comparisons among multiple groups,and Bonferroni’s post hoc test was applied for pairwise comparisons between groups.Results:1.Different Doses of UVB Exposure Induced Apoptosis in HaCaT Cells:Wild-type HaCaT cells were exposed to different doses of UVB（control,10,20,and30?mJ/cm²）for 24 hours.Compared with the Control group,cleaved PARP expression was significantly increased in all UVB-treated groups（P<0.05）,with the most pronounced change observed at 20?mJ/cm².Annexin V/PI double staining revealed a higher proportion of apoptotic cells following 20?mJ/cm²UVB exposure（P<0.05）,suggesting that UVB induces apoptosis in a dose-dependent manner,with 20?mJ/cm²potentially representing the optimal dose for inducing apoptosis.2.Different Doses of UVB Exposure Promote Nuclear Translocation of NRF1:Wild-type HaCaT cells were exposed to different doses of UVB（Control,10,20,and30?mJ/cm²）,and nuclear NRF1 protein levels were assessed by Western blot.Compared with the control group,nuclear NRF1 expression was significantly increased in all UVB-treated groups（P<0.05）,with the most marked upregulation observed at20?mJ/cm²（P<0.05）.3.NRF1 Silencing Enhances UVB-induced Apoptosis:Scramble and NRF1-KD HaCaT cells were treated with 20?mJ/cm²UVB.The results showed that cleaved PARP expression were significantly increased in NRF1-KD cells compared to Scramble（P<0.05）.These findings indicated that NRF1 silencing increased UVB-induced apoptosis.4.NRF1 Colocalizes with DNA Damage-associated Proteins:Wild-type HaCaT cells were exposed to UVB and analyzed by immunofluorescence to assess the spatial distribution of NRF1 with CPD andγ-H2AX.Following 20?mJ/cm²UVB treatment,NRF1 showed significant nuclear colocalization withγ-H2AX.Additionally,NRF1was colocalized with CPD-positive sites,suggesting that NRF1 may participate in the regulation of DNA repair by targeting DNA damage sites.5.UVB Exposure Induced Activation of Nuclear ATM and ATR Proteins:Western blot analysis of nuclear extracts from wild-type HaCaT cells showed that expression levels of ATR,phosphorylated ATR（p-ATR）,ATM and phosphorylated ATM（p-ATM）were significantly increased following 20?mJ/cm²UVB treatment（P<0.05）,indicating that UVB exposure induced activation of ATM and ATR in the nucleus.6.Potential Binding Sites between ATM,ATR and NRF1:Molecular docking demonstrated potential protein-protein interactions of NRF1 with ATM and ATR.7.Immunofluorescence Indicated Colocalization of NRF1 with ATM and ATR:Wild-type HaCaT cells were exposed to UVB and analyzed by immunofluorescence to assess the spatial distribution of NRF1 with ATM and ATR.Following 20?mJ/cm²UVB treatment,NRF1 showed significant nuclear colocalization with both ATM and ATR.8.Direct Protein Interactions Exist between NRF1 and ATM/ATR:In wild-type HaCaT cells exposed to 20?mJ/cm²UVB,co-immunoprecipitation assays revealed that NRF1 was detected in ATM-immunoprecipitated complexes,and ATM was detected in NRF1-immunoprecipitated complexes,indicating a direct interaction between ATM and NRF1.Similar co-immunoprecipitation results were observed for ATR and NRF1,suggesting a direct binding between ATR and NRF1 as well.9.Transfection ATR Mutant Rescued UVB-induced DNA Damage in NRF1-KD Cells:Studies have indicated that ATR is a direct responder to UVB exposure.Therefore,the phospho-mimetic ATR mutant（S428D）was transfected into NRF1-KD cells.Western blot analysis showed that p-ATR levels were significantly increased in NRF1-KD cells after mutant transfection（P<0.05）.Following 20?mJ/cm²UVB exposure,CPD fluorescence intensity was significantly higher in NRF1-KD cells compared to Scramble.However,compared with NRF1-KD cells transfected with control vector(ATR^vector),those transfected with the ATR mutant(ATR^mutant)exhibited a significant reduction in CPD levels（P<0.05）,indicating that sustained ATR phosphorylation can rescue the exacerbated DNA damage caused by NRF1 silencing.Conclusion:1.Nuclear translocation and activation of NRF1 protect against UVB-induced apoptosis.2.NRF1 directly interacts with ATM and ATR,and protected UVB-induced DNA damage by regulating ATR activity.更多还原