The Preliminary Study On The Pathologic Mechanism Of Acne Inversa Caused By NCSTN Gene-Notch Signaling Pathway

Objective:Acne inversa(AI;OMIM 142690),also known as Hidradenitis suppurativa(HS),is mainly caused by mutations of y-secretase gene,with the majority of NCSTN subunit mutations.In this study,a lentiviral vector-mediated RNA interference technique was used to construct a stable silenced the expression of NCSTN gene in human immortal keratinocyte cell line HaCaT.RNA sequencing method was used to detect the gene expression of the interference group and the negative control group.Then we screened differentially expressed signaling pathways,inflammatory factors and molecules involved in cell proliferation and differentiation by bioinformatics.After detected the changes in signaling pathways,inflammatory factors and proliferation and differentiation of keratinocytes,we made the preliminary exploration of the possible mechanism of NCSTN gene mutation causing familial AI.Methods:1.We Infected human immortal keratinocyte cell line HaCaT with a lentivirus-mediated short hairpin RNA(shRNA)expression plasmid targeting the NCSTN gene.These cells were divided into negative control group(NC group)and interference group(shRNA group).The transfection efficiency was detected by flow cytometry.The silence efficiency of NCSTN was detected by Real-Time PCR and Western blotting.2.RNA sequencing was used to detect the gene expression of HaCaT cells with stable silencing of NCSTN gene.Then we screened changed signaling pathways and molecules associated inflammatory,cell proliferation and differentiation by bioinformatics.3.Real-Time PCR and Western blotting were used to detect the expression of the four subunits of y-secretase and the change of Notch-Hes signaling pathway.4.Detection the expression of IL-36 cytokines(IL-36??IL-36??IL-36? and IL-36Ra).The expression of the downstream NF-?B pathway was detected in the same way.5.CCK-8 was used to detect cell proliferation,and Real-Time PCR and Western blotting were used to detect the expression of cytokeratins(CK1?CK5?CK7?CK10?CK14?CK16?CK17?CK18?CK19?CK20)and other differentiation factors(IVL?LOR).Results:1.After lenti-virus infected HaCaT cells,the transfection efficiency of NC group and shRNA group was greater than 90%by flow cytometry.Compared with NC group,the expression of NCSTN gene mRNA and protein in the interference group was significantly decreased.2.RNA sequencing revealed differential expression of multiple genes and altered signaling pathways.We selected Notch-Hes signaling pathway,IL-36 molecules and proliferation differentiation factors as targets of interest by bioinformatics.3.After the stable silence of NCSTN gene,the expression of NCSTN subunit,PSENEN subunit and PS1-CTF were significantly decreased compared with the control group,while the full length of PSEN subunit and APH1A subunit were unchanged.The corresponding expression of Notch3 and Hes1 was decreased.4.The levels of IL-36? and IL-36? were significantly increased,and the level of IL-36Ra was significantly decreased,while the levels of IL-36y were not significantly changed.There was no change in the expression of the downstream NF-?B pathway.5.The results of CCK-8 showed that the keratinocytes were proliferated actively in the interference group compared with the control group.The expression of cytokeratin CK16 and CK19 and terminal differentiation factor IVL were all significantly decreased,and the cells were abnormally differentiated.Conclusion:We made the preliminary exploration of the possible mechanism of NCSTN gene mutation causing familial AI.1.Human immortal keratinocyte model HaCaT with stable silencing of NCSTN gene was successfully constructed.2.RNA sequencing screened that the Notch signaling pathway was down-regulated,and IL-36 molecules and proliferation and differentiation factors changed.3.After the NCSTN gene silenced,the y-secretase-Notch-Hes signaling pathway is down-regulated.4.There was a imbalance of IL-36 cytokines after in the keratinocyte cells after NCSTN gene interference.The downstream NF-?B pathway is not changed.5.The keratinocytes showed hyperproliferation and abnormal differentiation after NCSTN gene was silenced.