中华皮肤科杂志 ›› 2022, Vol. 55 ›› Issue (2): 116-122.doi: 10.35541/cjd.20210346

• 论著 • 上一篇    下一篇

沉默信息调节因子1、3和缺氧诱导因子1α在皮肤鳞状细胞癌组织和细胞中的表达

葛新红1    焦亚宁 1    葛明昊2    马迎东2    石越2    王禹2    刘玲玲2   

  1. 1宁夏医科大学总医院皮肤科，银川  750004；2宁夏医科大学，银川  750004    
  • 收稿日期:2021-04-27 修回日期:2021-12-01 发布日期:2022-01-27
  • 通讯作者: 葛新红 E-mail:gexinhong.0101@163.com
  • 作者简介:年底结题需要该文刊出
  • 基金资助:
    宁夏回族自治区自然科学基金（2019AAC03178）；宁夏留学回国人员创新创业项目（2021-352）

Expression of silent information regulator 1 and 3 and hypoxia-inducible factor 1α in cutaneous squamous cell carcinoma tissues and cells

Ge Xinhong1, Jiao Yaning1, Ge Minghao2, Ma Yingdong2, Shi Yue2, Wang Yu2, Liu Lingling2   

  1. 1Department of Dermatology, General Hospital of Ningxia Medical University, Yinchuan 750004, China; 2Ningxia Medical University, Yinchuan 750004, China  
  • Received:2021-04-27 Revised:2021-12-01 Published:2022-01-27
  • Contact: Ge Xinhong E-mail:gexinhong.0101@163.com
  • Supported by:
    Natural Science Foundation of Ningxia Hui Autonomous Region of China（2019AAC03178）; Innovation and Entrepreneurship Project for Ningxia Returnees in 2021 （2021-352）

PDF

3

摘要： 【摘要】 目的 分析沉默信息调节因子1（Sirt1）、3（Sirt3）和缺氧诱导因子1α（HIF-1α）在皮肤鳞状细胞癌（CSCC）组织和细胞中的表达，探讨其在CSCC发生发展中的作用。方法 收集 2019年1月至2020年12月就诊于宁夏医科大学总医院皮肤科、经病理活检确诊为高、中、低分化CSCC患者的病变组织和非癌症患者正常皮肤组织各30份，同时培养CSCC细胞A431和HaCaT细胞。采用免疫组化、Western印迹和实时定量PCR（RT-PCR）分别检测Sirt1、Sirt3及HIF-1α蛋白和mRNA在不同分化程度CSCC和正常皮肤组织中的表达，用免疫荧光化学和RT-PCR法分别检测Sirt1、Sirt3及HIF-1α蛋白和mRNA在A431和HaCaT细胞中的表达。计量资料多组间均数比较采用单因素方差分析，两组间比较采用t检验。结果 免疫组化显示，Sirt3在正常皮肤和高、中、低分化CSCC中的表达水平（相对平均AOD值）分别为100 ± 12.12、117.72 ± 26.23、127.32 ± 24.45、132.71 ± 31.61，差异有统计学意义（F = 20.14，P < 0.001）；Sirt1和HIF-1α蛋白在正常皮肤组织及高、中、低分化CSCC中的表达亦呈逐渐增高趋势，且各组间差异均有统计学意义（F值分别为174.50和225.00，均P < 0.001）。Western印迹结果显示，Sirt3在正常皮肤及高、中、低分化CSCC中的表达水平（相对灰度值）分别为1.000 ± 0.132、1.403 ± 0.411、1.387 ± 0.393、1.677 ± 0.683，差异有统计学意义（F = 34.97，P < 0.001），Sirt1和HIF-1α的表达差异亦有统计学意义（F值分别为69.29和199.90，均P < 0.001），且具有逐渐升高的趋势。RT-PCR结果显示，Sirt3、Sirt1和HIF-1α mRNA在正常皮肤及高、中、低分化CSCC的表达逐渐增强，各组间差异有统计学意义（F值分别为113.00、174.50和50.33，均P < 0.001）。Sirt3、Sirt1和HIF-1α蛋白在A431细胞中的表达均高于HaCaT细胞（t值分别为16.75、18.34、27.76，均P < 0.001），mRNA表达亦均高于HaCaT细胞（t值分别为14.22、9.62、16.86，均P < 0.001）。结论 Sirt3、Sirt1和HIF-1α在CSCC组织和细胞中的表达增高，可能促进了CSCC的发生与发展。

关键词: 癌, 鳞状细胞, 细胞系, 肿瘤, 角蛋白细胞, 缺氧诱导因子1, α亚基, 沉默信息调节因子

Abstract: 【Abstract】 Objective To determine the expression of silent information regulator 1 （Sirt1）, Sirt3 and hypoxia-inducible factor 1α （HIF-1α） in cutaneous squamous cell carcinoma （CSCC） tissues and cells, and to explore their role in the occurrence and development of CSCC. Methods From January 2019 to December 2020, 30 lesional skin tissues were obtained from patients with histopathologically confirmed poorly-, moderately- or well-differentiated CSCC, and 30 normal skin tissues were obtained from patients with non-cancerous diseases in Department of Dermatology, General Hospital of Ningxia Medical University. A CSCC cell line A431 and a human keratinocyte cell line HaCaT were cultured. Immunohistochemical study, Western blot analysis and real-time quantitative PCR （RT-PCR） were performed to determine the protein and mRNA expression of Sirt1, Sirt3 and HIF-1α in CSCC tissues of different grades of differentiation and normal skin tissues, cytochemical and immunofluorescence staining and RT-PCR were conducted to determine the protein and mRNA expression of Sirt1, Sirt3 and HIF-1α in A431 and HaCaT cells, respectively. Comparisons of measurement data among multiple groups were performed by using one-way analysis of variance, and comparisons between two groups by using t test. Results Immunohistochemical study showed that the expression level of Sirt3 （expressed as the average optical density） was 100 ± 12.12, 117.72 ± 26.23, 127.32 ± 24.45, 132.71 ± 31.61 in the normal skin tissues and well-, moderately- and poorly-differentiated CSCC tissues respectively, and there was a significant difference among these groups（F = 20.14, P < 0.001）; the expression of Sirt1 and HIF-1α increased in turn from the normal skin tissues to the well-, moderately- and poorly-differentiated CSCC tissues, and significantly differred in these groups （F = 174.50, 225.00, respectively, both P < 0.001）. As Western blot analysis revealed, the expression level of Sirt3 significantly differed among the normal skin tissues, well-, moderately- and poorly-differentiated CSCC tissues （expressed as relative gray value: 1.000 ± 0.132, 1.403 ± 0.411, 1.387 ± 0.393, 1.677 ± 0.683, respectively; F = 34.97, P < 0.001）, and so did the expression levels of Sirt1 and HIF-1α （F = 69.29, 199.90, respectively, both P < 0.00l）, with a gradually increasing trend in their expression levels from the the normal skin tissues to well-, moderately- and poorly-differentiated CSCC tissues. RT-PCR showed that the mRNA expression of Sirt3, Sirt1 and HIF-1α was sequentially increased from the normal skin tissues to well-, moderately- and poorly-differentiated CSCC tissues, and significant differences were observed among these groups （F = 113.00, 174.50, 50.33, respectively, all P < 0.001）. The protein expression levels of Sirt3, Sirt1 and HIF-1α were significantly higher in the A431 cells than in the HaCaT cells （t = 16.75, 18.34, 27.76, respectively, all P < 0.001）, and so were their mRNA expression levels （t = 14.22, 9.62, 16.86, respectively, all P < 0.001）. Conclusion Increased expression of Sirt3, Sirt1 and HIF-1α was observed in CSCC tissues and cells， which may promote the occurrence and development of CSCC.

Key words: Carcinoma, squamous cell, Cell line, tumor, Keratinocytes, Hypoxia-inducible factor 1, alpha subunit, Silent information regulators