Research On The Changes Of Signal Pathway In Acne Inversa With PSENEN Gene Mutation

BackgroundsAcne Inversa(AI)is a chronic inflammatory skin disease of the hair folliclesebaceous gland-apocrine sweat gland unit.Skin lesions can represent abscesses,sinuses and scar,which affect patients' quality of life seriously.In 2010,Wang et al.first discovered that the mutation of ?-secretase gene was associated with the onset of AI,which was subsequently confirmed in different countries.?-secretase is a hotspot of familial AI,which consists of four components: presenilin(PS),nicastrin(NCT),and anterior pharynx defective-1(APH1),presenilin enhancer-2(PEN2).PSENEN gene mutations are found in many diseases,such as AI,Dowling-Degos disease(DDD),familial acne,Alzheimer's disease,but the role of PSENEN gene in skin diseases is still not clear.ObjectivesThe aim of this study was to investigate the effects of PSENEN knockdown on ?-secretase in keratinocytes and changes in related signaling pathways,as well as,on the biological formation of keratinocytes such as proliferation,differentiation and apoptosis.At the same time,to explore the pathogenesis and therapeutic targets of AI PSENEN gene mutation patients,trying to find a unique molecular mechanism that different from Alzheimer's.Methods(1)Design three siRNA sequences specifically knocking down PSENEN,and divide the cultured human immortalized keratinocyte cell line(HaCaT cells)into experimental group(PSENEN-siRNA),negative control group(m-siRNA),blank control group(Add equal amount of transfection reagent),verify the interference efficiency by real-time quantitative PCR and Western blotting,and construct a PSENEN knockdown model of HaCaT cells.(2)Whole transcriptome sequencing was used to detect the difference expression of mRNA in PSENEN knockdown HaCaT cells.The effects of PSENEN on cells were analyzed by GO analysis and KEGG analysis.(3)Real-time quantitative PCR and Western blotting was used to verify the different signaling pathway,the effect on ?-secretase function,and the effect on the genes related to proliferation and differentiation.CCK-8 was used to detect cell proliferation and flow cytometry for apoptosis,in the aspect of cell behavioral changes.(4)Six AI patients with PSENEN gene mutation and six normal control skins were collected.Immunohistochemistry was applied to confirmed the changes of mTOR signaling pathways.Results(1)The expression of PEN2 mRNA and protein in HaCaT cells was down-regulated by PSENEN for 72h(p< 0.05)so that the PSENEN knockdown cell model was successfully constructed.Compared with the control,?-secretase components changed with imNCT increased,mNCT decreased,PS1-CTF decreased,and PS1 increased(p < 0.05),while the APH1 a remained unchanged,in the experimental group.(2)Transcriptome sequencing results showed that most of the differential biological functions of the mRNA were related to metabolism.Most of the molecular functions were related to the intermolecular binding.In KEGG analysis(2 fold difference,p< 0.05),the expression of the EGFR,VEGFR,mTOR signaling pathway is elevated,while decreased in the expression of insulin,m-TOR and metabolic signaling pathways.The metabolic pathways of carbohydrates,lipids and amino acids in the metabolic pathways all decreased,especially decreased in glucose metabolism.(3)There was no significant difference in Ki67 mRNA and protein levels,CCK-8 and apoptotic.The expression of EGFR mRNA and protein were increased(p < 0.05),but the PI3K-AKT-mTOR signaling pathway related protein was not statistically significant.(4)The expression of mTOR was significantly increased in 6 patients and mTOR was highly expressed in the epidermis,follicular infundibulum,appendage and inflammatory cells.Conclusions(1)The PSENEN knockdown of HaCaT cells decreased the expression of PEN2 protein resulting in the ?-secretase NCT maturation disorder and PS1 phosphorylation disorder.PSENEN knockdown caused a decrease in cellular metabolic levels and elevation of the EGFR signaling pathway.(2)mTOR is widely expressed in skin lesions of all PSENEN mutation patients and its activation may be associated with AI pathogenesis,so that it might become a new target for treatment in the future.