中华皮肤科杂志 ›› 2020, Vol. 53 ›› Issue (6): 445-451.doi: 10.35541/cjd.20190957

• 论著 • 上一篇    下一篇

nicastrin基因沉默的HaCaT细胞基因表达谱分析

肖学敏1    何艳艳2    徐浩翔2    王宝玺3    林立航1   

  1. 1福建医科大学附属协和医院皮肤科，福州  350001；2中国医学科学院  北京协和医学院  皮肤病医院，南京  210042；3中国医学科学院  北京协和医学院  整形外科研究所皮肤科，北京  100144
  • 收稿日期:2019-10-08 修回日期:2020-04-07 发布日期:2020-06-01
  • 通讯作者: 王宝玺；林立航 E-mail:wangbx@ncstdlc.org; 460879404@qq.com
  • 基金资助:
    国家自然科学基金（81602785）；福建省自然科学基金（2017J05128）；福建省卫生健康中青年骨干人才培养项目（2019-ZQN-47）；福建中医药大学科研平台校管课题（X2018018-平台）

Genome-wide expression profile analysis of Nicastrin gene-silenced HaCaT cells

Xiao Xuemin1, He Yanyan2, Xu Haoxiang2, Wang Baoxi3, Lin Lihang1   

  1. 1Department of Dermatology, Fujian Medical University Union Hospital, Fuzhou 350001, China; 2Hospital for Skin Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China; 3Department of Dermatology, Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100144, China
  • Received:2019-10-08 Revised:2020-04-07 Published:2020-06-01
  • Contact: Wang Baoxi; Lin Lihang E-mail:wangbx@ncstdlc.org; 460879404@qq.com
  • Supported by:
    National Natural Science Foundation of China（81602785）; Fujian Province Natural Science Foundation （2017J05128）; Fujian Provincial Health Technology Project （2019-ZQN-47）; The Opening Foundation of Research Platform of Fujian University of Traditional Chinese Medicine （X2018018-platform）

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摘要： 【摘要】 目的 通过沉默人永生化角质形成细胞HaCaT细胞中nicastrin（NCSTN）基因的表达，研究其下游细胞增殖及分化相关信号通路的改变。方法 将HaCaT细胞分为干扰组、阴性对照组和空白对照组：干扰组转染特异性NCSTN-siRNA，阴性对照组转染阴性对照siRNA，空白对照组转染等量转染试剂。实时PCR及Western印迹检测各组NCSTN mRNA和蛋白表达验证转染效率。运用Agilent人类全基因组表达谱芯片技术研究干扰组HaCaT细胞基因表达谱与阴性对照组之间的差异，以表达上调或者下调倍数 ≥ 2.0且P ≤ 0.05为标准，将差异基因用GO分析进行富集，筛选出表达差异显著且与角质形成细胞增生分化相关的基因，用实时PCR验证结果。结果 干扰组HaCaT细胞NCSTN mRNA及蛋白相对表达量分别为0.287 ± 0.090、0.443 ± 0.085，明显低于阴性对照组（0.969 ± 0.127、1.047 ± 0.114）以及空白对照组（1.000 ± 0.151、1.000 ± 0.111），差异均有统计学意义（F值分别为30.787、31.139，P值均为0.001）。表达谱芯片显示，与阴性对照组相比，干扰组表达下调基因605条，上调基因444条。GO分析显示，干扰后差异表达基因富集到上皮发育、上皮细胞分化、角质形成细胞分化、角化4种生物学过程。对表达差异显著且与角质形成细胞增生分化相关的Sprouty相关蛋白2基因、表皮生长因子7基因、胰岛素样生长因子结合蛋白5基因、人Rho关联含卷曲螺旋蛋白激酶2基因和骨形成发生蛋白6基因通过实时PCR验证，验证结果与芯片结果显示的差异趋势一致。结论 NCSTN基因功能缺失有可能通过调节其下游细胞增殖及分化相关信号通路的表达，影响角质形成细胞的正常增殖和分化。

关键词: 化脓性汗腺炎, 角蛋白细胞, 基因表达谱, 细胞增殖, 细胞分化, Nicastrin基因

Abstract: 【Abstract】 Objective To investigate changes of nicastrin （NCSTN） downstream molecules in signaling pathways related to cell proliferation and differentiation after silencing the expression of the NCSTN gene in the human immortalized keratinocyte cell line HaCaT. Methods HaCaT cells were divided into 3 groups: interference group transfected with a specific small interfering RNA （siRNA） targeting NCSTN （NCSTN-siRNA）, negative control group transfected with a negative control siRNA, and blank control group transfected with the equal amount of transfection reagent. Real-time PCR and Western blot analysis were performed to measure the NCSTN mRNA and protein expression in groups, in order to verify the transfection efficiency. Differences in gene expression profiles in HaCaT cells were detected between the interference group and negative control group by using Agilent whole-genome microarray, and differentially expressed genes were identified based on a fold change ≥ 2.0 with a P value ≤ 0.05. Gene ontology（GO） enrichment analysis was employed to identify the roles of the differentially expressed genes, and then to screen out significantly differentially expressed genes associated with proliferation and differentiation of keratinocytes, some of which were verified by real-time PCR. Results The interference group showed significantly decreased mRNA and protein expression of NCSTN （0.287 ± 0.090, 0.443 ± 0.085, respectively） compared with the negative control group （0.969 ± 0.127, 1.047 ± 0.114, respectively） and blank control group （1.000 ± 0.151, 1.000 ± 0.111, F = 30.787, 31.139, respectively, both P = 0.001）. Whole genome-expression analysis using an Agilent microarray platform revealed 605 downregulated genes and 444 upregulated genes in HaCaT cells in the interference group compared with the negative control group. GO analysis showed that differentially expressed genes were enriched into 4 biological processes, including epithelial development, epithelial cell differentiation, keratinocyte differentiation and keratinization. The significantly differentially expressed genes associated with proliferation and differentiation of keratinocytes, including the Sprouty-related protein with EVH1 domain 2, fibroblast growth factor 7, insulin-like growth factor-binding protein 5, Rho-associated coiled-coil kinase 2 and bone morphogenetic protein 6 genes, were verified by real-time PCR, and the verification results were consistent with the difference trend shown by the microarray results. Conclusion The loss of NCSTN gene function may affect the normal proliferation and differentiation of keratinocytes by regulating the expression of its downstream molecules in signaling pathways associated with cell proliferation and differentiation.

Key words: Hidradenitis suppurativa, Keratinocytes, Gene expression profiling, Cell proliferation, Cell differentiation, Nicastrin gene

引用本文

肖学敏 何艳艳 徐浩翔 王宝玺 林立航. nicastrin基因沉默的HaCaT细胞基因表达谱分析[J]. 中华皮肤科杂志, 2020,53(6):445-451. doi:10.35541/cjd.20190957

Xiao Xuemin, He Yanyan, Xu Haoxiang, Wang Baoxi, Lin Lihang. Genome-wide expression profile analysis of Nicastrin gene-silenced HaCaT cells[J]. Chinese Journal of Dermatology, 2020, 53(6): 445-451.doi:10.35541/cjd.20190957