| BackgroundScar formation is an unavoidable and very difficult issue in the process of wound healing.After severe skin injuries such as deep burns,scar formation presents as various degrees,which seriously affects the appearance and function of the involved site.The prevention and treatment of hypertrophic scars is one of the important fields of clinical researches.Unfortunately,until now the mechanism of the formation and development of hypertrophic scars is not yet very clear,which results in the limited therapeutic stratagies and effects.It is found that fibroblasts are one of the main functional cells involved in wound healing.It plays an vital role in the development of hypertrophic scars by synthesizing excessive extracellular matrix(ECM)and hyperproliferation.TGF-?1 is a key factor in the process of tissue fibrosis.Many studies have shown that TGF-?1 and its downstream target molecules affect tissue fibrosis by affecting cell proliferation,apoptosis,differentiation,autophagy and local immune response.In the early stage of tissue repair,TGF-?1 is highly expressed in hypertrophic scars,which is closely related to the formation of scars.Our research team found that TGF-?1 can promote P311 gene expression in dermal fibroblasts,and P311 plays an important role in tissue fibrosis and fibroblast to myofibroblast transformation.In addition,P311 It is also involved in many important pathophysiological processes,such as nerve regeneration,tumor invasion,blood pressure homeostasis,blood vessel regeneration,alveologenesis and so on.However,the mechanism of P311 upstream regulation is not clear.The process of wound repair includes three steps: inflammation,hyperplasia and remodeling.Platelet degranulation is responsible for the release and activation of a series of effective cytokines,including epidermal growth factor(EGF),transforming growth factor ?1(TGF-?1),which are used as chemoattractants to recruit macrophages,neutrophils,epithelial cells and fibroblasts.In normal wounds,apoptosis and extracellular matrix(ECM)remodeling mediate the biosynthesis and degradation of new tissues.In the process of excessive scar formation,the dysfunction of potential regulatory mechanism may lead to persistent inflammation,excessive collagen synthesis or insufficient matrix degradation and remodeling.TGF-?1 plays an important role in these regulatory mechanisms.Therefore,in-depth understanding of the effect of TGF-?1 on the transcription and expression of fibroblasts can help us to further clarify the mechanism of TGF-?1 in the process of fibrosis.At the same time,it also provides some clues for us to study how TGF-?1 regulates P311 expression.Part 1 Prediction and identification of P311 gene promoter regionMethods:1.Prediction of P311 gene promoter position by BioinformaticsThe human cell epigenetics database Roadmap was used to analyze the epigenetic modification levels in different regions of the potential promoter of the P311 gene,including H3K4me1,H3K4me2,H3K4me3,and DNase.2.Construction of P311 Promoter Truncated PlasmidThe promoter fragment was amplified by PCR,the size of the fragment was identified by agarose gel,the p GL3-Basic vector and the target fragment were ligated by double digestion,and then plasmid synthesis and sequencing were used to identify the plasmid synthesis.3.Detection of promoter activity and identification of active promoter regionsThe target plasmid was transfected into 293 T cells using Lip2000,and the activity of promoters of different fragment sizes(promoter-1 series and promoter-1 series)was detected using the dual luciferase reporting system.Results:1.Prediction of P311 gene promoter position by BioinformaticsWe selected two potential promoter sequences,named promoter-1(chr5: 111092954-111094954)and promoter-2(chr5: 111333162-111335162)(hg38 as the reference genome).The level of epigenetic modification of the sequence was analyzed through the Roadmap database,and the results showed that the enrichment level of H3K4me3 and DNase on the promoter-1 sequence was higher than that of promoter-2.2.Construction of P311 Promoter Truncated PlasmidWe successfully constructed series pGL3-Basic plasmids of several truncated promoter-1,including 2kb,1.5kb,1kb,and 0.6kb sizes;uccessfully constructed series pGL3-Basic plasmids of several truncated promoter-1,including,1.5kb,1kb,0.8kb,0.6kb,0.4kb,and 0.2kb sizes.3.Detection of promoter activity and identification of active promoter regionsWe used dual Luciferase Report System to identify plasmids with different fragment sizes.The results showed that promoter-2 had promoter characteristics.pro-0.4 is the core promoter of P311 gene.Part 2 Study on the Transcriptional Regulation Mechanism of TGF-?1 on P311 GeneMethods1.Effect of TGF-?1 stimulation on transcriptome expression of fibroblastsIllumina sequencing was used to detect the expression and differences of transcripts in fibroblasts stimulated by TGF-?1.The differential genes were analyzed by GO and KEGG to find enriched signal pathways and biological functions;2.Effect of TGF-?1 on P311 gene transcriptionReal-time quantitative PCR was used to detect the difference in mRNA expression levels of P311 gene before and after TGF-?1 stimulation;3.Transcriptional regulation of meox1 on P311Using Jaspar database to predict the potential transcription factors binding to P311 core promoter,we constructed meox1 eukaryotic overexpression plasmid and specific interfering RNA.After transfection,P311 in meox1 overexpression group,meox1 interfering group and control group were detected by real-time fluorescent quantitative PCR MRNA expression level;construction of pro-0.4 plasmid with meox1 binding site deletion,detection of promoter activity difference between the deletion group and the control group by double luciferase reporting system;TGF-? 1 stimulation of cells,detection of meox1 protein concentration in P311 promoter region by chromatin immunoprecipitation fluorescence quantitative PCR in the stimulation group and the control group.4.Meox1 protein expression in human hypertrophic scar specimensFrom January 2018 to June 2019,3 cases of hypertrophic scar patients(2 males and 1 female,aged 35 to 56)were treated in the First Affiliated Hospital of the Army Military Medical University(Third Military Medical University)8 to 12 months after burns.Aged),the scar tissue and normal skin tissue attached to the scar edge were taken for immunohistochemical staining,and the expression and distribution of Meox1 protein was observed.Results:1.Effect of TGF-?1 stimulation on transcriptome expression of fibroblastsGO analysis showed that the effects of TGF-?1 on skin fibroblasts mainly involved processes such as tissue repair,cell migration and chemotaxis induction of inflammatory cells;KEGG pathway analysis showed that TGF-?1 may play a role in skin fibroblasts It is through TNF,IL-17 or extracellular matrix receptors and other potential signaling pathways.TGF-?1 stimulated fibroblasts had higher levels of metabolism and transcription than controls.2.Effect of TGF-?1 on P311 gene transcriptionIn HDF-a cells,TGF-?1 can significantly promote the expression level of P311 m RNA.3.Transcriptional regulation of meox1 on P311We used JASPAR database to predict the potential transcription factors that can be used to bind to pro-2-4,including ZNF384,TFAP2 C,Meox1,MZF1,and YY1;Meox1 overexpression can increase the expression level of P311 mRNA.The expression level was significantly reduced;the deletion of the binding site of Meox1 in the P311 promoter region significantly inhibited the activity of the P311 promoter;TGF-?1 stimulation increased Meox1's enrichment in the P311 promoter region.4.Meox1 protein expression in human hypertrophic scar specimensMeox1 protein expression in scar tissue of patients with hypertrophic scar was significantly higher than that of normal skin tissue.Part 3 Study on the mechanism of TGF-?1 regulating Meox1 and the effect of Meox1 on the biological function of fibroblastsMethod1.Effect of TGF-?1 stimulation on Smad2/3/4 protein enrichment in Meox1 promoter regionChIP-q PCR was used to detect the enrichment of Smad2,Smad3,and Smad4 proteins in the Meox1 promoter region in the TGF-?1 stimulation group and the stimulation group,respectively.2.Effect of TGF-?1 on P311 gene transcriptionFirst,we constructed a Smad2/3/4 eukaryotic overexpression plasmid and synthesized Smad2/3/4-specific interfering RNA,then transfected the plasmid or interfering RNA with Lip2000 into HDF-a cells,and then detected Meox1 by RT-q PCR.At the same time,the ChIP-q PCR was used to detect the changes of Smad2/3/4 overexpression and knockdown,and its protein enrichment in the Meox1 promoter region.3.Transcriptional regulation of Meox1 on P311Wound-healing assays and Transwell assays were used to detect the effect of Meox1 knockdown or overexpression on HDF-a cell migration ability;CCK8 cytotoxicity assays were used to detect the effect of Meox1 knockdown or overexpression on HDF-a cell proliferation ability,respectively.Results1.Effect of TGF-?1 stimulation on Smad2/3/4 protein enrichment in Meox1 promoter regionTGF-?1 stimulation can up-regulate the enrichment of Smad2,Smad3 and Smad4 proteins in the Meox1 promoter region2.Effect of Smad2/3 protein interference and overexpression on Meox1 promoter activitySmad2,Smad3,and Smad4 protein overexpression can significantly increase the transcription level of Meox1 m RNA;after Smad2,Smad3,and Smad4 protein overexpression,the enrichment of Smad2 and Smad3 in Meox1 is higher than that in the control group,while Smad4 and the control group have no significant difference;After interference or overexpression of Smad2,Smad3,Smad4,significant changes in the degree of enrichment of Smad2 and Smad3 in the promoter region of Meox1 gene were observed,but Smad4 did not change significantly.3.Effects of Meox1 on the migration and proliferation of HDF-a cells.Wound-healing assays results showed that the cell healing width in the Meox1 overexpression group was larger than that in the control group,and the cell healing width in the Meox1 knockdown group was smaller than that in the control group.The knockdown group had significantly fewer migrating cells than the control group.ConclusionIn this study,by constructing a truncated plasmid and performing dual luciferase system experiments,the promoter of the P311 gene was identified for the first time at chr5: 111999065-111999465(hg38 as the reference genome).And through transcriptome sequencing,promoter transcription factor analysis and chromatin immunoprecipitation experiments,it was confirmed that TGF-?1 acts on human dermal fibroblasts by regulating the homeobox gene 1(Meox1)and then transcriptionally regulates the P311 gene.Expression,which in turn affects its migration.In this paper,through RNA interference and overexpression experiments,it was confirmed that TGF-?1 mainly regulates the transcription level of Meox1 gene by affecting the enrichment of transcription factors Smad2 and Smad3 in the promoter region of Meox1 gene.In summary,this study found that TGF-?1 transcriptionally regulates the expression of Meox1 by affecting the enrichment of Smad2 and Smad3 in the promoter region of the Meox1 gene,and then affects the activity and transcription level of the P311 gene promoter,thereby affecting fibroblast migration.And the ability to multiply. |
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