中华皮肤科杂志 ›› 2023, Vol. 56 ›› Issue (8): 724-736.doi: 10.35541/cjd.20220905

• 论著 • 上一篇    下一篇

Brahma相关基因1与激活转录因子2相互作用对皮肤鳞状细胞癌细胞增殖、迁移和侵袭能力的影响

张俐1    施健1    葛鑫2    刘念念1    陈赛1    张冬梅2    缪旭1   

  1. 1南通大学第二附属医院皮肤科，南通  226001；2南通大学第二附属医院临床医学研究中心，南通  226001
  • 收稿日期:2022-12-21 修回日期:2023-06-13 发布日期:2023-08-07
  • 通讯作者: 缪旭 E-mail:mx730129@163.com
  • 基金资助:
    江苏省自然科学基金（BK20211108）；南通市民生科技计划项目（MS22022046）；南通市卫生健康委员会科研立项课题（MB2021019）

Effects of the interaction between Brahma-related gene 1 and activating transcription factor 2 on the proliferation, migration and invasion of cutaneous squamous cell carcinoma cells

Zhang Li1, Shi Jian1, Ge Xin2, Liu Niannian1, Chen Sai1, Zhang Dongmei2, Miao Xu1   

  1. 1Department of Dermatology, Affiliated Hospital 2 of Nantong University, Nantong 226001, Jiangsu, China; 2Medical Research Center, Affiliated Hospital 2 of Nantong University, Nantong 226001, Jiangsu, China
  • Received:2022-12-21 Revised:2023-06-13 Published:2023-08-07
  • Contact: Miao Xu E-mail:mx730129@163.com
  • Supported by:
    Natural Science Foundation of Jiangsu Province of China （BK20211108）; Nantong Livelihood Science and Technology Program （MS22022046）; Nantong Health Commission Scientific Research Project （MB2021019）

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摘要： 【摘要】 目的 探讨Brahma相关基因1（BRG1）在皮肤鳞状细胞癌（cSCC）组织及细胞中的表达，及其与激活转录因子2（ATF2）相互作用调控cSCC细胞增殖、迁移、侵袭的分子机制。方法 收集2015—2021年南通大学第二附属医院皮肤科经病理确诊为日光性角化病（AK）、cSCC（含原位鳞癌）患者的石蜡组织标本分别66例和80例，同时收集皮肤美容手术修整下的正常皮肤组织石蜡标本35例作为正常对照组。采用免疫组化染色法检测BRG1在cSCC、AK及正常皮肤组织中的表达，分析BRG1表达与cSCC患者临床病理参数之间的相关性。收集cSCC患者和健康对照新鲜组织标本各12例，常规培养cSCC细胞株A431和Scl-1及人永生化角质形成细胞株HaCaT，实时荧光定量PCR（qRT-PCR）检测组织及细胞中BRG1 mRNA的表达，通过免疫共沉淀和细胞免疫荧光染色分析BRG1与ATF2的相互作用。通过RNA干扰法敲低A431、Scl-1细胞中BRG1（BRG1 siRNA1 ~ 5组）和ATF2表达（ATF2-shRNA组），并分别转染阴性对照siRNA或shNC作为对照（control siRNA组、shNC组），采用细胞计数（CCK8）实验、克隆形成实验、细胞划痕实验和Transwell实验分别检测敲低BRG1、ATF2对cSCC细胞增殖、迁移及侵袭的影响。多组间计量资料的比较采用单因素方差分析，两两多重比较采用Dunnett-t检验。结果 免疫组化染色显示，BRG1蛋白在cSCC、AK组织中的表达强度显著低于正常皮肤组织（χ2 = 44.40，P < 0.001）。qRT-PCR显示，cSCC组织中BRG1 mRNA表达水平（1.345 ± 0.956）显著低于正常皮肤组织（2.499 ± 1.501，t = 2.25，P = 0.035），A431、Scl-1细胞中BRG1 mRNA表达水平（0.041 ± 0.002、0.026 ± 0.003）亦显著低于HaCaT细胞（0.135 ± 0.033，t = 4.95、5.73，P = 0.008、0.005）。BRG1的低表达与cSCC患者癌组织位于曝光部位（P = 0.041）、肿瘤低分化程度（P = 0.001）、Broder高分级（P < 0.001）有关。BRG1 siRNA1组和BRG1 siRNA2组A431、Scl-1细胞克隆形成数、迁移细胞数、侵袭细胞数及细胞迁移率均显著高于control siRNA组（均P < 0.05）。免疫共沉淀实验表明，在A431、Scl-1细胞中BRG1蛋白与ATF2蛋白能相互结合，免疫荧光法显示二者存在共定位；与control siRNA组相比，A431（BRG1 siRNA1、2组）和Scl-1细胞（BRG1 siRNA1组）中敲低BRG1表达可促进ATF2磷酸化激活（均P < 0.05）；与shNC组相比，shATF2组A431、Scl-1细胞克隆形成数（均P = 0.001）、24 ~ 96 h细胞增殖活性（均P < 0.001）、迁移细胞数及侵袭细胞数均显著降低（均P ≤ 0.001）。结论 BRG1在cSCC及AK组织中低表达，且BRG1可抑制cSCC细胞增殖、迁移及侵袭；ATF2促进cSCC细胞增殖、迁移及侵袭；BRG1可能通过与ATF2蛋白相互作用并抑制ATF2磷酸化激活，从而发挥抑癌作用。

关键词: 癌, 鳞状细胞, 皮肤, 角化病, 光化性, Brahma相关基因1, 激活转录因子2, SWI/SNF复合体, 染色质重塑

Abstract: 【Abstract】 Objective To determine the expression of Brahma-related gene 1 （BRG1） in cutaneous squamous cell carcinoma （cSCC） tissues and cells, and to investigate molecular mechanisms underlying the regulatory effect of its interaction with activating transcription factor 2 （ATF2） on the proliferation, migration and invasion of cSCC cells. Methods From 2015 to 2021, 66 paraffin-embedded actinic keratosis （AK） tissue samples and 80 paraffin-embedded cSCC （including squamous cell carcinoma in situ） tissue samples were collected from the Department of Dermatology, Affiliated Hospital 2 of Nantong University, and the diagnoses of all the cases were confirmed histopathologically; at the same time, 35 paraffin-embedded normal skin tissue samples obtained by cosmetic surgery served as normal control group. Immunohistochemical staining was performed to determine the BRG1 expression in cSCC, AK, and normal skin tissues, and correlations between BRG1 expression and clinicopathological parameters of cSCC patients were analyzed. Fresh tissue samples were collected from 12 cSCC patients and 12 healthy controls, and cSCC cell lines A431 and Scl-1 and a human immortalized keratinocyte cell line HaCaT were routinely cultured; real-time fluorescence-based quantitative PCR （qRT-PCR） was performed to determine the mRNA expression of BRG1 in tissues and cells, and co-immunoprecipitation assay and cellular immunofluorescence staining were conducted to analyze the interaction between BRG1 and ATF2. The expression of BRG1 （BRG1 siRNA1 - 5 groups） and ATF2 （ATF2-shRNA group） in A431 and Scl-1 cells was knocked down by RNA interference, and cells transfected with negative control siRNA or shNC served as controls （control siRNA group and shNC group, respectively）, cell counting kit-8 （CCK8） assay, colony formation assay, cell scratch assay, and Transwell assay were conducted to evaluate effects of knocking down BRG1 and ATF2 on the proliferation, migration, and invasion of cSCC cells. Comparisons of measurement data among multiple groups were conducted using one-way analysis of variance, and multiple comparisons were conducted using Dunnett-t test. Results Immunohistochemical staining showed that the expression intensity of BRG1 protein was significantly lower in the cSCC and AK tissues than in the normal skin tissues （χ2 = 44.40, P < 0.001）. qRT-PCR showed that the mRNA expression level of BRG1 was significantly lower in the cSCC tissues （1.345 ± 0.956） than in the normal skin tissues （2.499 ± 1.501, t = 2.25, P = 0.035）, and also significantly lower in A431 and Scl-1 cells （0.041 ± 0.002, 0.026 ± 0.003, respectively） than in HaCaT cells （0.135 ± 0.033, t = 4.95, 5.73, P = 0.008, 0.005, respectively）. The low expression of BRG1 was associated with tumors at sun-exposed sites （P = 0.041）, low tumor differentiation （P = 0.001）, and high Broder′s grade （P < 0.001） in the cSCC patients. In both A431 cells and Scl-1 cells, the BRG1 siRNA1 group and BRG1 siRNA2 group showed significantly increased numbers of cell colonies, migratory cells and invasive cells, as well as cell migration rates compared with the control siRNA group （all P < 0.05）. Co-immunoprecipitation assay showed that BRG1 protein could bind to ATF2 protein in A431 and Scl-1 cells, and immunofluorescence staining showed that the two proteins were co-localized; compared with the control siRNA group, the BRG1 siRNA1 group （both A431 and Scl-1 cells） and BRG1 siRNA2 group （A431 cells） both showed increased phosphorylation and activation of ATF2 （all P < 0.05）; in both A431 cells and Scl-1 cells, the shATF2 group showed significantly decreased numbers of cell colonies （both P = 0.001）, cellular proliferative activity at 24 - 96 hours （all P < 0.001）, and numbers of migratory cells and invasive cells compared with the shNC group （all P ≤ 0.001）. Conclusion BRG1 was lowly expressed in the cSCC and AK tissues, and could inhibit the proliferation, migration, and invasion of cSCC cells; ATF2 could promote the proliferation, migration, and invasion of cSCC cells; BRG1 may exert an anti-tumor effect by interacting with ATF2 protein and inhibiting phosphorylation-dependent activation of ATF2.

Key words: Carcinoma, squamous cell, Skin, Keratosis, actinic, BRG1, ATF2, SWI/SNF complex, Chromatin remodeling

引用本文

张俐 施健 葛鑫 刘念念 陈赛 张冬梅 缪旭. Brahma相关基因1与激活转录因子2相互作用对皮肤鳞状细胞癌细胞增殖、迁移和侵袭能力的影响[J]. 中华皮肤科杂志, 2023,56(8):724-736. doi:10.35541/cjd.20220905

Zhang Li, Shi Jian, Ge Xin, Liu Niannian, Chen Sai, Zhang Dongmei, Miao Xu. Effects of the interaction between Brahma-related gene 1 and activating transcription factor 2 on the proliferation, migration and invasion of cutaneous squamous cell carcinoma cells[J]. Chinese Journal of Dermatology, 2023, 56(8): 724-736.doi:10.35541/cjd.20220905