Objective:Acne inversa is a multifactorial chronic debilitating disease.Genetic factors are involved in 40%of patients,especially the nicastrin(NCSTN)gene.However,the role of the mutated NCSTN gene in the pathogenesis...Objective:Acne inversa is a multifactorial chronic debilitating disease.Genetic factors are involved in 40%of patients,especially the nicastrin(NCSTN)gene.However,the role of the mutated NCSTN gene in the pathogenesis of acne inversa remains unclear.Retinoic acid is recommends to treat moderate to severe acne inversa,therefor we conduct this in vitro research to study the association between NCSTN gene mutation and the retinoic acid signaling pathway in human immortalized skin keratinocyte(HaCaT)cells.Methods:HaCaT cells were infected with a lentivirus-mediated short hairpin RNA(shRNA)expression plasmid specifically targeting the NCSTN gene.Real-time polymerase chain reaction(PCR)and Western blotting were used to detect the interference efficiency of NCSTN.RNA sequencing was used to detect differential genes in the NSCTN-deficient HaCaT cells.Based on bioinformatics analysis and clinical treatment data,the retinoic acid signal pathway was selected for screening.Quantitative PCR was used to verify the changes in the expressions of retinoic acid signaling pathway-related receptors and molecules in the HaCaT cell line after NCSTN silencing.The Student t test and one-way analysis of variance were used to evaluate intergroup differences.Results:Sequencing showed that the NCSTN-shRNA lentiviral recombinant expression plasmid was successfully constructed.After lentivirus infection of HaCaT cells,real-time PCR results showed significantly reduced NCSTN mRNA expression in the interference group compared with the negative control group,and the interference efficiency was 75.0%.Western blotting showed that the inhibition rate of NCSTN protein expression in the shRNA group was 71.7%.RNA sequencing revealed significant differential expression of some genes,and changes in signaling pathways.Compared with the control group,the group with the silenced NCSTN showed significantly decreased expression of retinoic acid receptors(RARα:F=23.482,RARβ:F=603.241,RXRα:F=69.689,and RARRES1:F=167.482,and all P<0.001),and peroxisome proliferator-activated receptorγ(F=8.138,P<0.01).Conclusion:Defective function of the NCSTN gene leads to an impaired retinoic acid signaling pathway in HaCaT cells,which suggests that the retinoic acid signaling pathway may play a role on the onset of acne inversa caused by NCSTN gene mutation.展开更多
目的探讨Nicastrin(NCSTN)基因启动子区多态性与散发阿尔茨海默病(sporadic Alzheimer s dis-ease,SAD)发病的关系及其机制。方法用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)和直接测序的方法对中国北方汉族359例SAD患者和331名正...目的探讨Nicastrin(NCSTN)基因启动子区多态性与散发阿尔茨海默病(sporadic Alzheimer s dis-ease,SAD)发病的关系及其机制。方法用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)和直接测序的方法对中国北方汉族359例SAD患者和331名正常对照者NCSTN基因启动子区单核苷酸多态性位点(SNP)进行筛查,基因分型后,进行病例对照-相关分析。构建三种不同基因型启动子质粒,通过双荧光报告基因系统检测不同启动子质粒的转录活性。结果①中国北方人群中的NCSTN基因启动子区ATG上游2245 bp范围内存在3个SNP:-1216C/A(rs2147471),-796T/G(rs10752637)和-436C/T(rs1324738);②-1216C/A和-796T/G的基因型频率在各自的SAD组和正常对照组中的分布差异有统计学意义;③三种启动子质粒的转录活性差异无统计。结论NCSTN启动子区SNP:-796T/G和-1216C/A,与SAD的发病具有相关性,但未发现这两个位点具有病理学功能。展开更多
基金funded by the National Natural Science Foundation of China(No.81472872)the CAMS Innovation Fund for Medical Sciences(No.2016-I2M-1-002)the Central Universities Fundamental Research Funds in PUMC(No.3332019160).
文摘Objective:Acne inversa is a multifactorial chronic debilitating disease.Genetic factors are involved in 40%of patients,especially the nicastrin(NCSTN)gene.However,the role of the mutated NCSTN gene in the pathogenesis of acne inversa remains unclear.Retinoic acid is recommends to treat moderate to severe acne inversa,therefor we conduct this in vitro research to study the association between NCSTN gene mutation and the retinoic acid signaling pathway in human immortalized skin keratinocyte(HaCaT)cells.Methods:HaCaT cells were infected with a lentivirus-mediated short hairpin RNA(shRNA)expression plasmid specifically targeting the NCSTN gene.Real-time polymerase chain reaction(PCR)and Western blotting were used to detect the interference efficiency of NCSTN.RNA sequencing was used to detect differential genes in the NSCTN-deficient HaCaT cells.Based on bioinformatics analysis and clinical treatment data,the retinoic acid signal pathway was selected for screening.Quantitative PCR was used to verify the changes in the expressions of retinoic acid signaling pathway-related receptors and molecules in the HaCaT cell line after NCSTN silencing.The Student t test and one-way analysis of variance were used to evaluate intergroup differences.Results:Sequencing showed that the NCSTN-shRNA lentiviral recombinant expression plasmid was successfully constructed.After lentivirus infection of HaCaT cells,real-time PCR results showed significantly reduced NCSTN mRNA expression in the interference group compared with the negative control group,and the interference efficiency was 75.0%.Western blotting showed that the inhibition rate of NCSTN protein expression in the shRNA group was 71.7%.RNA sequencing revealed significant differential expression of some genes,and changes in signaling pathways.Compared with the control group,the group with the silenced NCSTN showed significantly decreased expression of retinoic acid receptors(RARα:F=23.482,RARβ:F=603.241,RXRα:F=69.689,and RARRES1:F=167.482,and all P<0.001),and peroxisome proliferator-activated receptorγ(F=8.138,P<0.01).Conclusion:Defective function of the NCSTN gene leads to an impaired retinoic acid signaling pathway in HaCaT cells,which suggests that the retinoic acid signaling pathway may play a role on the onset of acne inversa caused by NCSTN gene mutation.
文摘目的探讨Nicastrin(NCSTN)基因启动子区多态性与散发阿尔茨海默病(sporadic Alzheimer s dis-ease,SAD)发病的关系及其机制。方法用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)和直接测序的方法对中国北方汉族359例SAD患者和331名正常对照者NCSTN基因启动子区单核苷酸多态性位点(SNP)进行筛查,基因分型后,进行病例对照-相关分析。构建三种不同基因型启动子质粒,通过双荧光报告基因系统检测不同启动子质粒的转录活性。结果①中国北方人群中的NCSTN基因启动子区ATG上游2245 bp范围内存在3个SNP:-1216C/A(rs2147471),-796T/G(rs10752637)和-436C/T(rs1324738);②-1216C/A和-796T/G的基因型频率在各自的SAD组和正常对照组中的分布差异有统计学意义;③三种启动子质粒的转录活性差异无统计。结论NCSTN启动子区SNP:-796T/G和-1216C/A,与SAD的发病具有相关性,但未发现这两个位点具有病理学功能。