Effects Of Increased KRT17 Expression Under High-Glucose On Keratinocytes And Dermal Fibroblasts

Objective: The main aim of this study was to investigate the effects of overexpressed keratin 17(KRT17)on the biology of human immortalized keratinocytes(HaCaT)and human dermal fibroblasts(HDFs)under pathological diabetic conditions,including cell proliferation,migration,and collagen synthesis,and to study the effect and mechanism of KRT17 on diabetic wound healing.Methods: It was found in the RNA sequencing(RNA-seq)result of our previous study that high glucose can stimulate three types of human skin cells,namely human epidermal keratinocytes(HEKs),human dermal fibroblasts(HDFs),and human dermal microvascular endothelial cells(HDMECs)to express KRT17 m RNA,mainly expressed in HEK.First,the expression of KRT17 was performed in diabetic keratinocytes(HEKs and HaCaTs),diabetic animal models(db/db diabetic mice and HFD/STZ-induced diabetic mice),and diabetic skin tissues using real-time quantitative fluorescence polymerase chain reaction(RT-q PCR),western blotting,ELISA,and tissue immunohistochemistry(IHC).Subsequently,HaCaTs amd HDFs stimulated with different concentrations of KRT17 were established in vitro.Changes in HaCaT and HDF cell proliferation and migration were observed using CCK8 cell proliferation detection,cell wound scratch assay,and transwell migration assay.Next,RNA sequencing(RNA-seq)was performed.The sequencing results were analyzed using differential gene expression,Gene Ontology(GO)functional significant enrichment,and KEGG pathway significant enrichment analyses.According to the KEGG analysis results,the key signaling pathways and molecules of KRT17 acting on HaCaTs and HDFs were screened and verified in the cell model.Based on the influence of KRT17 on HDF collagen synthesis in RNA-seq results,changes in collagen synthesis in HDFs stimulated by KRT17 were detected using RT-q PCR and western blotting in vitro.In addition,Masson staining and collagen content detection were performed on diabetic skin tissue to observe changes in dermal collagen.Results: KRT17 expression was upregulated in HEKs and HaCaTs after high glucose stimulation.KRT17 expression was upregulated in skin of db/db diabetic mice,HFD/STZinduced diabetic mice,and patients with diabetes.In vitro stimulation of HaCaTs with different concentrations of KRT17 resulted in increased cell proliferation and cell migration.The RNA-seq data showed 493 differentially expressed genes(DEGs)were identified.Among them,297 genes were upregulated and 196 were downregulated.GO analysis showed that most of the enriched gene composition of the plasma membrane,as well as the regulation of transcriptional adhesion and inflammation.KEGG analysis revealed activation of the c-MYb/PI3K-Akt signaling pathway involved in regulating cell proliferation and migration,and it was validated in vitro by KRT17-stimulated HaCaT.In vitro stimulation of HDFs with different concentrations of KRT17 resulted in no significant changes in cell proliferation but inhibition of cell migration.The RNA-seq data showed 537 differentially expressed genes(DEGs)were identified.Among them,244 genes were upregulated and 293 were downregulated.GO analysis showed that most of the enriched genes were related to ECM synthesis and regulation.The pathway with the largest number of genes enriched in KEGG analysis was the phosphatidylinositol 3?kinase/protein kinase(PI3K-Akt)signaling pathway,in which integrin alpha-11(ITGA11)m RNA,a key molecule that regulates cell migration,was significantly downregulated.ITGA11 expression is downregulated after stimulation of HDFs by KRT17 in vitro.In vitro stimulation with KRT17 inhibited collagen synthesis in HDFs.Masson staining of the diabetic skin tissue revealed dermal collagen deposition disorder and decreased collagen content.Conclusions: Under pathological diabetes mellitus,the expression of KRT17 in keratinocytes of skin tissue increases.The increased KRT17 can promote the proliferation and migration of keratinocytes through the c-MYB /PI3K-AKT pathway.What is more,the increased KRT17 parocrine action on fibroblasts inhibits the migration of fibroblasts by downregulating the expression of ITGA11 in fibroblasts and by inhibiting the synthesis of collagen in HDFs.The increased expression of KRT17 in high glucose pathology can affect skin function by regulating the function of skin cells,such as wound healing.KRT17 may become a molecular target for the treatment of diabetic wound healing...