| Chronic actinic dermatitis(CAD)is a refractory,recurrent,immune-mediated photosensitive skin disease.Clinical manifestations are infiltrating erythema,papules or plaques at exposure sites,which sometimes present diffuse thickening,and in severe cases can transform into cutaneous T-cell lymphoma,which seriously affects physical and mental health and quality of life of patients.Recent reports show that with the increase of climate change and environmental pollution,the incidence of chronic actinic dermatitis is on the rise,which can account for 7%-17%of the global photosensitive diseases,while domestic studies show that CAD can account for the top three photosensitive diseases.At present,the pathogenesis of CAD is still not explained clearly.Studies at home and abroad suggested that the incidence of CAD may be related to oxidative stress,abnormal proliferation and apoptosis,immune imbalance and UV mediated delayed hypersensitivity reaction.CD4+invasion was dominant in early CAD lesions,while CD8+invasion was dominant in late CAD lesions.Meanwhile,the ratio of Th2/Th1 in peripheral blood of patients was unbalanced or inverted,and was positively correlated with the severity of disease.Therefore,immune abnormalities play a very important role in the pathogenesis of CAD.Studies have shown that keratinocytes in the epidermis can express many pattern recognition receptors,which are distributed under the tight junction barrier.When the tight junction is damaged,foreign antigens will be recognized by pattern recognition receptors through the damaged tight junction,and then lead to abnormal immune response of the skin.CAD mostly occurs in farmers or middle-aged and elderly men who love outdoor sports,presenting as invasive erythema and most of the patients have substance allergy.So,does CAD also has barrier damage?Are immune abnormalities in CAD related to barrier damage?It has not been reported.In order to further explore the pathogenesis of CAD,our team used the second-generation high-throughput sequencing to perform transcriptome sequencing on CAD skin lesions at early stage,screened and analyzed differentially expressed mRNA.The results showed that CLDN1,CLDN5 and OCLN were differentially low expressed.So,are these gene expression differences also reduced at protein level?And how are they regulated?It has not been reported.Through bioinformatic analysis,it was found that c-Fos(AP-1 subunit)was bound to the promoter regions of CLDN1,CLDN5 and OCLN,and c-Fos showed significant low expressed in transcriptome sequencing results.Further,bioinformatics analysis showed that RP11-35612.4 was co-localized and co-expressed with c-Fos(RP 11-35612.4 showed significant low expressed in transcriptome sequencing results).Then,does RP11-35612.4 interact with c-Fos to regulate the expression of CAD barrier genes?It has not been reported yet.The research content of this paper includes the following two parts:Part Ⅰ Protein profile and transcriptome analysis of chronic actinic dermatitisObjective:Proteins in CAD lesion and normal tissues were detected by 4D labeled free quantitative proteomics,and differentially expressed proteins were screened for correlation analysis with transcriptome.Methods:The exposed skin lesions of 4 patients with CAD and normal tissues of 4 healthy controls were collected,then proteins were extracted,and differentially expressed proteins in CAD skin lesions were screened out by liquid chromatography mass spectrometry,and further bioinformatics analysis was conducted.RNA was extracted from extra lesions in the above tissues(3 CAD tissues and 3 normal tissues),and transcriptome sequencing was performed to screen differentially expressed mRNA.Finally,the correlation between differentially expressed protein and mRNA was analyzed.Results:1.The concentration of extracted proteins from collected tissues met the requirements for quality control,and OD values of extracted RNA were all between 1.8-2.2,which met the experimental requirements for second-generation high-throughput sequencing.2.The results of quantitative proteomics showed that there were significant differences in protein expression between CAD tissue and healthy controls.A total of 341 proteins were up-regulated and 238 were down-regulated(P<0.05).Transcriptomic sequencing results showed that 745 mRNA were up-regulated and 602 mRNA were down-regulated(P<0.05).3.GO(Gene Ontology)enrichment analysis,KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis,and protein structural domain enrichment were analyzed,results showed that immune-related pathways and barrier damage were significantly enriched.4.Bioinformatics prediction showed that c-Fos could bind to the promoter regions of CLDN1,CLDN5 and OCLN.Conclusion:Through analyzing differentially expressed proteins and mRNA,it was found that differentially expressed genes in CAD tissues were enriched in immune-related pathways and barrier damage pathways.Among barrier related genes,c-Fos was obvious low expressed which has a regulatory relationship with CLDN1,CLDN5 and OCLN,and c-Fos is co-localized and co-expressed with RP11-35612.4.Part Ⅱ LncRNA-RP11-356I2.4 affects skin permeability barrier through recruiting transcription factor c-FosObjective:To verify the differential expression of barrier protein(CLDN1,CLDN5 and OCLN)and RNA(c-Fos,RP11-35612.4)in CAD tissue,and to explore whether RP11-356I2.4 affects the expression of barrier genes by regulating c-Fos transcription factor and the relationship between RP11-356I2.4 and ultraviolet.Moreover,to observe the repair effect of RP 11-35612.4 on UV-induced barrier injury model in mice.Methods:1.Noninvasive skin barrier detection was performed on lesion of CAD and healthy people.2.The expression of CLDN1,CLDN5 and OCLN in CAD patients and healthy controls were detected by immunohistochemistry.3.si-RP11-356,2.4(silence,si),si-RP11-NC(negative control,NC),OE-RP11-356I2.4(over expression,OE),OE-RP11-NC,and si-Fos,si-Fos-NC,OE-Fos,OE-Fos-NC,etc.were artificially synthesized and transfected into HaCaT cells.RT-qPCR was used to detect the efficiency of knockdown or overexpression.4.After knockdown of RP 11-35612.4,transepithelial electric resistance(TEER)of cells was detected by electrometer,and leakage of FITC-Dextran was detected by microplate reader.5.After knockdown or overexpression of RP11-356I2.4,expressions of CLDN1,CLDN5 and OCLN were detected by RT-qPCR and western blot.6.Expression of c-Fos and RP11-356I2.4 in CAD and healthy people were detected by RT-qPCR;c-Fos expression was detected after knockdown and overexpression of RP11-356I2.4.7.After c-Fos knockdown and overexpression,expressions of CLDN1,CLDN5 and OCLN were detected by RT-qPCR and western blot.8.After c-Fos overexpression,the binding of c-Fos to CLDN1,CLDN5 and OCLN promoter regions was detected by CHIP-PCR.Meanwhile,the promoter reporter gene assay was used to detect the activity of CLDN1,CLDN5 and OCLN promoters after overexpression of RP11-356I2.4 or c-Fos.9.After overexpression of RP11-356I2.4,c-Fos and Jun were extracted from RP11-356I2.4 probe by RNA pull down assay.10.Leakage of FITC-Dextran and TEER value were detected after irradiation of keratinocytes with different doses of UVB(0,5,10,20,30mJ/cm2)and UVA(0,0.5,1,2,4J/cm2).11.Cells were exposed to ultraviolet UVB(30mJ/cm2)and UVA(4J/cm2),and samples were taken at multiple time points to detect the expression of RP11-356I2.4 by RT-qPCR.12.To detect the methylation ratio of RP11-356I2.4 promoter region in CAD.At the same time,the methylation ratio of RP11-356I2.4 promoter region was detected after UVB irradiation in keratinocytes.13.These barrier genes were detected after knockdown of RP 11-356I2.4 or knockdown of RP11-356I2.4+ultraviolet radiation on keratinocytes.14.Solar simulator was used to irradiate mouse skin to make UV-induced barrier damage model,and the expression of RP11-356I2.4 was detected by RT-qPCR.15.Adenovirus-coated RP11-356I2.4 was injected into the back skin of mice,and then non-invasive skin barrier detection was performed on mice.At the same time,the expressions of CLDN1,CLDN5 and OCLN were detected by immunohistochemistry.Results:1.Compared with control group,TEWL in lesion of CAD was significantly increased while SCH was significantly decreased.2.The expressions of CLDN1,CLDN5 and OCLN in CAD tissue were all decreased.3.After RP11-356I2.4 knockdown,TEER value decreased significantly,while leakeage of FITC-Dextran increased significantly.4.After RP11-356I2.4 knockdown,the results of RT-qPCR showed that CLDN1,CLDN5,FLG,LOR and ZO-1 were all decreased.At the same time,after knockdown of RP11-356I2.4,western blot showed that the expression of CLDN1,CLDN5 and OCLN were all decreased,while overexpression of RP11-356I2.4 could be partially reversed.5.The expression of RP11-356I2.4 and c-Fos in patient tissues was decreased,and both of them was positively correlated.RT-qPCR assay showed that RP11-356I2.4 could positively regulate c-Fos expression.6.After c-Fos knockdown,the results of RT-qPCR showed that CLDN1,CLDN5,IVL,ZO-1 and OCLN were all decreased.Meanwhile,after c-Fos knockdown,the protein expression of CLDN1,CLDN5 and OCLN was decreased,while overexpression of c-Fos could be partially reversed.7.After c-Fos overexpression,CHIP-PCR detection showed that c-Fos could bind to the promoter regions of CLDN1,CLDN5 and OCLN.The promoter reporter assay showed c-Fos activating the promotor activity of CLDN1,CLDN5 and OCLN.8.Overexpression of RP11-356I2.4 could enhance the luciferase activity of promoter reporter gene,while the luciferase activity decreased after addition of AP-1 blocker.9.After overexpression of RP11-356I2.4,c-Fos and c-Jun can be pulled down from the probes targeting RP11-356I2.4.10.UV induced keratinocyte permeability barrier damage.11.Ultraviolet radiation can induce a decrease in the expression of RP11-356I2.4 within a certain period of time(6h-24h),and UVB is more obvious than UVA.12.The promoter methylation ratio of RP 11-356I2.4 was increased in CAD;Meanwhile,UVB irradiation can induce an increase in the methylation ratio of promoter region of RP11-356I2.4 in keratinocytes.13.The low expression of RP 11-356I2.4 leads to decreased cell tolerance to ultraviolet.14.The expression of RP11-356I2.4 was decreased in UV-induced barrier damage model mice.Injecting adenovirus-coated RP11-35612.4 into mouse skin can repair the UV-induced barrier damage in mouse models.Conclusion:Patients with CAD had significant skin barrier damage.RP 11-356I2.4 not only positively regulates the expression of c-Fos,but also regulates the promoter region of barrier genes through recruiting c-Fos(AP-1)transcription factor.In addition,UV irradiation can increase the methylation ratio of CpG site in the promoter region of RP11-356I2.4.Adenovirus-coated RP11-356I2.4 can repair the UV-induced barrier injury model in mice. |
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