| Objective:The study aims to explore the relationship between NCSTN gene and acne inversa,and explore the specific mechanism of keratinocyte proliferation,differentiation and inflammation when the NCSTN gene silencing.Methods:The study used lentivirus-mediated RNA interference technology to construct the HaCaT cell model of human immortalized keratinocytes with stable NCSTN gene interference.RNA sequencing and TMT protein labeling methods were used to detect the differential genes in the HaCaT cell model with stable NCSTN gene interference and the differently expressed genes were verified.Through bioinformatics analysis combined with clinical data,screening retinoic acid signaling pathway,NF-κB signaling pathway related molecules and so on for verification research.1.Infect the human immortalized keratinocyte HaCaT cells with a lentiviral vector-mediated short hairpin RNA expression plasmid targeting the NCSTN gene.Cells were divided into blank group,negative control group and interference group.Real-Time PCR and Western blot were used to detect the interference efficiency of NCSTN gene.Combining TMT-labeled proteomics and RNA sequencing technology to analyze the HaCaT cells with stable knockdown of the NCSTN gene,the mRNA levels of the top10 differentially expressed genes with a common expression trend were verified.2.Using RNA high-throughput sequencing technology and TMT marker quantitative proteomics technology to jointly analyze the mRNA level and protein expression in HaCaT cells with stable interference of NCSTN gene,and screen out the two detection technologies that have a common expression trend Real-Time PCR method was used to verify the mRNA expression of the first 10 genes.3.Through bioinformatics analysis and combined with clinical data,screen and detect retinoic acid signaling pathway related molecules(RARα,RARβ,RARγ,RXRα,RXRβ,RXRγ,PPARγ,RARRES1,RARRES2),and NF-κB signaling pathway(NFKB1 NFKB2,REL,NFKBIA)mRNA expression.Results:1.After infection with HaCaT cells by lentivirus,compared with the negative control group,Real-Time PCR results showed that the NCSTN mRNA expression in the interference group was significantly reduced(P<0.001),and the interference efficiency reached 75.0%.The results of Western blot showed that the inhibition rate of NCSTN protein expression in the shRNA group was 71.7%.After analyzing the human immortalized keratinocyte HaCaT with stable interference of NCSTN gene using TMT-labeled quantitative proteomics technology,we found that the number of differentially expressed proteins was 23,and 12 of these differentially expressed proteins were down-regulated and 11 were up-regulated.GO enrichment analysis showed that the functions with the most clustering among the gene attributes were:melanosomes,pigment particles,thin film rafts,thin film regions,etc.2.After using RNA sequencing technology and TMT-labeled quantitative proteomics technology for joint analysis,we found differential expression of multiple genes and changes in multiple signaling pathways.Real-Time PCR results of the first 9 genes that were jointly up-regulated and the first 10 genes that were jointly down-regulated in the two detection technologies showed that the mRNA expression levels of the genes LRBA,DSG3,SNX2,OAS3,ATP2B1,LZIC,VSNL1,UQCRFS1 were up-regulated,Consistent with the sequencing results;mRNA expression levels of genes KIAA0020,C15orf52,NCSTN,PSEN2,LACTB,DNTTIP2,CDH2,CDH4,SCAF1,IDH1 were up-regulated,consistent with sequencing results.3.After stable interference of NCSTN gene,the expressions of RARα,RARβ,RXRα,RARRES1 related to retinoic acid signaling pathway were significantly decreased compared with the control group(p<0.001),PPARy was significantly decreased(p<0.01),RARγ,RXRβ,The difference between RXRy and RARRES2 was not significant and had no statistical significance.The expressions of NF-κB1,NF-κB2,and REL related to NF-κB signaling pathway were significantly reduced(P<0.001),and the differential expression of NFKBIA gene was not significant.Conclusion:The study explored the effect of NCSTN gene function defects on HaCaT cells.Using TMT proteomics technology and RNA high-throughput sequencing technology for joint analysis and verification,it was initially confirmed that NCSTN gene function defects may cause damage to retinoic acid signaling pathway and NF-κB signaling pathway in keratinocytes,combined with the use of tretinoin in AI patients The clinical treatment of drugs is effective,which further proves that the retinoic acid signaling pathway is involved in the occurrence and development of AI. |
