肿瘤坏死因子受体相关蛋白1基因沉默CD24^-CD44^+人喉鳞癌干细胞生物学特性的改变  

TRAP1 gene silencing effect on biological properties of CD24^-CD44^+ human laryngeal squamous cell carcinoma stem cells

作　　者：苏静[1] 薛海涛[1] 田君海[1] 张继华[1] Su Jing Xue Hai-tao Tian Jun-hai Zhang Ji-hua(Department of ENT, the First Hospital of Hebei Medical University, Shijiazhuang 050000, Hebei Province, Chin)

机构地区：[1]河北医科大学第一医院耳鼻喉科,河北省石家庄市050000

出　　处：《中国组织工程研究》2017年第5期663-668,共6页Chinese Journal of Tissue Engineering Research

基　　金：河北省重点研发计划自筹项目(152777179)~~

摘　　要：背景:肿瘤坏死因子受体相关蛋白1的异常表达与多种肿瘤的发生、发展密切相关。因此靶向抑制肿瘤坏死因子受体相关蛋白1的表达成为治疗或干预肿瘤生长的重要靶点。目的:观察肿瘤坏死因子受体相关蛋白1基因沉默对人喉鳞癌干细胞生长及凋亡的影响。方法:采用流式细胞技术特异性分离出CD24-CD44+喉鳞癌干细胞。设计及合成肿瘤坏死因子受体相关蛋白1的si RNA序列,脂质体TM2000转染CD24-CD44+喉鳞癌干细胞。流式细胞仪、MTT实验、细胞克隆形成实验、TUNEL凋亡实验评价沉默肿瘤坏死因子受体相关蛋白1基因对CD24-CD44+喉鳞癌干细胞增殖、凋亡的影响。结果与结论:(1)相关因子表达:相比于CD24+CD44-的人喉鳞癌细胞,CD24-CD44+人喉鳞癌干细胞内干性基因OCT4,SOX2,NANOG,肿瘤坏死因子受体相关蛋白1的表达均上调(P<0.05)。RNA干扰后,肿瘤坏死因子受体相关蛋白1的表达显著降低(P<0.05);(2)细胞周期及增殖:肿瘤坏死因子受体相关蛋白1基因沉默后人喉鳞癌干细胞的生长速度明显减慢(P<0.05),细胞停滞在G0/G1期,在S期细胞数减少(P<0.05),M期细胞无明显变化。肿瘤坏死因子受体相关蛋白1基因沉默后人喉鳞癌干细胞的增殖受到明显的抑制(P<0.05);(3)细胞克隆形成能力:相比于未经转染的人喉鳞癌干细胞,肿瘤坏死因子受体相关蛋白1基因沉默后人喉鳞癌干细胞克隆形成能力显著下调(P<0.05);(4)细胞凋亡:相比于未经转染的人喉鳞癌干细胞,肿瘤坏死因子受体相关蛋白1基因沉默后人喉鳞癌干细胞的凋亡基因BAD及BAX表达上调(P<0.05);(5)结果表明,特异性干扰肿瘤坏死因子受体相关蛋白1基因表达可抑制人喉鳞癌干细胞增殖并促进其凋亡。BACKGROUND： Studies have indicated that the abnormal expression of tumor necrosis factorreceptor-associated protein 1（TRAP1） is closely related to the occurrence and development of a variety of tumors. Therefore, targeted inhibition of TRAP1 expression has become an important target for the treatment or intervention of tumor growth. OBJECTIVE： To explore the effect of the TRAP1 gene silencing on the proliferation and apoptosis of human laryngeal squamous cell carcinoma stem cells. METHODS： CD24-CD44-human laryngeal squamous cell carcinoma stem cells were isolated by flow cytometry. Interfering RNA（si RNA） sequences for small molecule TRAP1 gene was designed and transferred into human laryngeal cancer stem cells by LipofectamineTM 2000. Flow cytometry, MTT assay, cell clone formation assay and TUNEL apoptosis assay were used to evaluate the effect of silencing TRAP1 gene on the proliferation and apoptosis of CD24-CD44＋ laryngeal cancer stem cells. RESULTS AND CONCLUSION： Compared with CD24＋CD44-cells, CD24-CD44＋ cells upregulated OCT4, SOX2, NANOG and TRAP1 expression levels（P〈0.05）. However, the expression of TRAP1 protein in human laryngeal squamous cell carcinoma was significantly decreased after RNA interference（P〈0.05）. The growth rate of TRAP1 gene silenced human laryngeal squamous cell carcinoma was significantly reduced（P〈0.05）, the cell arrest was in the G0/G1 phase, the number of cells in the S phase was decreased（P〈0.05）, and there was no significant change in the M phase. TRAP1 gene silencing significantly inhibited the proliferation of human laryngeal squamous cell carcinoma stem cells（P〈0.05）. Compared to the non-transfected cells, the TRAP1 gene silencing significantly reduced the clone formation ability of transfected human laryngeal squamous cell carcinoma stem cells（P〈0.05）, and TRAP1 gene silenced-human laryngeal squamous cell carcinoma stem cells were more easy to trigger apoptosis by upregulating BAD and BAX expression levels（P〈0.05

关 键 词：喉肿瘤 肿瘤干细胞 细胞增殖 细胞凋亡 RNA 小分子干扰 干细胞 肿瘤坏死因子受体相关蛋白1 人喉鳞癌 化疗耐药 基因沉默 细胞数量 克隆形成 组织工程 

分 类 号：R394.2[医药卫生—医学遗传学]