国际口腔医学杂志

国际口腔医学杂志 ›› 2019, Vol. 46 ›› Issue (6): 631-639.doi: 10.7518/gjkq.2019096

• 论著 • 上一篇    下一篇

锌指蛋白32在口腔鳞状细胞癌中的表达意义及对口腔鳞状细胞癌干细胞的影响

陈宏丽1,杨敬2,尹刚2,李皓缘3,乔燕4()   

  1. 1. 山西医科大学汾阳学院口腔医学教研室 汾阳 032000
    2. 山西医科大学附属汾阳医院口腔科 汾阳 032200
    3. 山西医科大学口腔医学院 太原 030001
    4. 铜川市人民医院南院口腔科 铜川 727031
  • 收稿日期:2019-01-26 修回日期:2019-07-26 出版日期:2019-11-01 发布日期:2019-11-14
  • 通讯作者: 乔燕 E-mail:2335878508@qq.com
  • 作者简介:陈宏丽,讲师,硕士,Email： ddd4782@163.com
  • 基金资助:
    山西省卫生和计划生育科研课题(20150183)

Expression of zinc finger protein 32 in oral squamous cell carcinoma and its effect on oral squamous cell carcinoma stem cells

Chen Hongli1,Yang Jing2,Yin Gang2,Li Haoyuan3,Qiao Yan4()   

  1. 1. Dept. of Stomatology and Teaching Research, Fenyang College of Shanxi Medical University, Fenyang 032000, China
    2. Dept. of Stomatology, Fenyang Hospital Affiliated to Shanxi Medical University, Fenyang 032200, China
    3. School of Stomatology, Shanxi Medical University, Taiyuan 030001, China
    4. Dept. of Stomatology, South Hospital of Tongchuan People’s Hospital, Tongchuan 727031, China;
  • Received:2019-01-26 Revised:2019-07-26 Online:2019-11-01 Published:2019-11-14
  • Contact: Yan Qiao E-mail:2335878508@qq.com
  • Supported by:
    This study was supported by Shanxi Health Family Planning Research Project(20150183)

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摘要： 目的 研究锌指蛋白32（ZNF32）在口腔鳞状细胞癌（OSCC）组织中的表达及与OSCC患者临床病例特征的关系,并探讨ZNF32对肿瘤干细胞（CSC）生物学特性的影响。 方法 反转录、定量即时聚合酶链反应（qRT-PCR）检测ZNF32 mRNA在45例OSCC组织和15例正常口腔黏膜组织中的表达水平,并分析OSCC组织中ZNF32的表达与临床病例特征的关系。磁珠分选OSCC细胞系Cal-27中的CSC。Western-blot检测有机阳离子/肉毒碱转运蛋白4（OCT4）、Nanog同源框（Nanog）、性别决定区Y框蛋白2（SOX2）干性标志蛋白及ZNF32在CSC中的表达;经脂质体分别转染ZNF32 siRNA（si-ZNF32）和对照（si-NC）48 h后,3-(4,5-二甲基吡啶-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四唑（MTS）增殖实验检测各组CSC的增殖能力,平板克隆检测各组CSC克隆形成能力,Transwell实验检测各组CSC转移能力,Western-blot检测各组CSC中信号传导转录激活因子3（STAT3）和磷酸化的信号传导转录激活因子3（pSTAT3）蛋白的表达。 结果 qRT-PCR结果显示ZNF32 mRNA在OSCC组织中的表达显著高于正常口腔黏膜组织,其高表达与OSCC肿瘤低分化、TNM分期晚期及淋巴结转移显著相关（P< 0.05）。OCT4、Nanog、SOX2干性标志蛋白在CSC中表达显著增加;ZNF32在CSC中的表达均显著高于OSCC细胞Cal-27和人口腔黏膜角化（HOK）细胞（P<0.05）;转染ZNF32 siRNA后,CSC细胞增殖、平板克隆形成及转移能力均下降,CSC细胞中pSTAT3蛋白的表达下降（P<0.05）。 结论 ZNF32在OSCC组织和细胞系中均高表达,且高表达与肿瘤低分化、TNM分期晚期及淋巴结转移相关,同时干扰ZNF32的表达抑制OSCC的CSC生物学特性,ZNF32可以作为治疗OSCC的潜在分子靶点。

关键词: 口腔鳞状细胞癌, 锌指蛋白32, 肿瘤干细胞, 信号传导转录激活因子3

Abstract: Objective To investigate the expression of zinc finger protein 32 (ZNF32) in oral squamous cell carcinoma (OSCC) and the relationship between the expression of ZNF32 in OSCC and the clinical features, and to explore the effect of ZNF32 on OSCC cancer stem cells (CSCs).Methods Quantitative real time polymerase chain reaction (qRT-PCR) was used to detect the expression of ZNF32 mRNA in 45 OSCC tissues and 15 normal oral mucosa tissues. The relationship between the expression of ZNF32 in OSCC tissues and clinical features was analyzed. Magnetic beads were used to sort CSCs in the OSCC cell line Cal-27. Western-blot was used to detect the expression of organic cation/carnitine transporter 4 (OCT4), Nanog homeobox (Nanog), sex determining region Y-box2 (SOX2) stem marker protein and ZNF32 in CSCs. After transfected with ZNF32 siRNA (si-ZNF32) and control (si-NC) for 48 h,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was used to detect the proliferation, plate clones were used to detect the ability of CSCs to form clones. Transwell assay was used to detect the metastasis ability of CSCs. Western-blot was used to detect the expression of signal transducer and activator of transcription 3 (STAT3) and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) proteins in CSCs.Results The expression of ZNF32 mRNA in OSCC tissues was significantly higher than that in normal oral mucosa. The high expression of ZNF32 mRNA was significantly correlated with OSCC tumor low differentiation, advance TNM stage and lymph node metastasis (P<0.05). The expression of OCT4, Nanog and SOX2 stem marker proteins was significantly increased in CSCs. The expression of ZNF32 in CSCs was significantly higher than that in OSCC cells Cal-27 and human oral keratinocyte (P<0.05). After transfection with ZNF32 siRNA, the cell proliferation, plate clone formation and metastasis ability of CSCs decreased, and the expression of pSTAT3 protein in CSCs cells decreased (P<0.05).Conclusion ZNF32 is highly expressed in OSCC tissues and cell lines, and high expression is associated with low tumor differentiation, advanced TNM stage and lymph node metastasis. Interfering with the expression of ZNF32 and inhibits the biological characteristics of OSCC CSCs. ZNF32 can be used as a potential treatment molecular targets for OSCC.

Key words: oral squamous cell carcinoma, zinc finger protein 32, cancer stem cell, signal transducer and activator of transcription 3

中图分类号: 

  • R739.85

图 1

qRT-PCR检测ZNF32 mRNA在OSCC组织中的表达水平 A：ZNF32 mRNA在OSCC组织中的相对表达量显著高于正常口腔黏膜组织;B：ZNF32 mRNA在低分化组织中的相对表达量显著高于高分化组织;C：ZNF32 mRNA在TNM分期Ⅲ~Ⅳ期组织中的相对表达量显著高于Ⅰ~Ⅱ期组织; D：ZNF32 mRNA在有淋巴结转移的组织中相对表达量显著高于无淋巴结转移组织。*：P<0.05。"

表 1

ZNF32 mRNA在OSCC组织中的表达水平与临床病例特征之间的关系"

临床病例特征 例数 ZNF32 mRNA相对表达量 t值 P值
性别 男 28 1.30±0.25 1.327 0.545
女 17 1.17±0.18
年龄/岁 <55 15 1.23±0.24 0.984 0.084
≥55 30 1.27±0.20
肿瘤大小/cm <4 36 1.20±0.50 3.048 0.061
≥4 9 1.45±0.33
组织学分级 高分化 26 1.06±0.40 3.445 0.001
低分化 19 1.50±0.46
TNM分期 Ⅰ~Ⅱ 23 1.02±0.36 3.613 0.000
Ⅲ~Ⅳ 22 1.49±0.48
淋巴结转移 无 23 1.01±0.30 3.869 0.000
有 22 1.50±0.51

图 2

Western-blot检测OCT4、Nanog和SOX2干性标志蛋白的表达"

"

图 4

MTS检测ZNF32对CSC增殖能力的影响*：P<0.05。"

"

"

图 7

Western-blot检测ZNF32对STAT3信号通路的影响"

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