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

doi:10.3969/j.issn.2095-4344.2017.05.002

Abstract

Abstract:

BACKGROUND: Studies have indicated that the abnormal expression of tumor necrosis factor receptor-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 (siRNA) sequences for small molecule TRAP1 gene was designed and transferred into human laryngeal cancer stem cells by Lipofectamine^TM 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). Overall, our experimental results indicate that the specific interference of TRAP1 gene expression could inhibit the proliferation and promote apoptosis of human laryngeal squamous cell carcinoma stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Laryngeal Neoplasms, Neoplastic Stem Cells, Cell Proliferation, Apoptosis, RNA, Small Interfering

CLC Number:

R394.2

Su Jing, Xue Hai-tao, Tian Jun-hai, Zhang Ji-hua. TRAP1 gene silencing effect on biological properties of CD24-CD44+ human laryngeal squamous cell carcinoma stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(5): 663-668.

Figures/Tables 1

2.1 人喉鳞癌干细胞的分离及筛选体外培养人喉鳞癌细胞，待细胞处于生长对数期时，胰酶消化为单细胞生长体系，流式细胞筛选出CD24-CD44+的人喉鳞癌细胞。为进一步鉴定上述细胞的干细胞特性，采用Western blot实验检测了与干性相关基因(OCT4，SOX2及NANOG)的表达。结果显示，相比于CD24+CD44-的人喉鳞癌细胞，CD24-CD44+人喉鳞癌细胞内OCT4上调(9.0±1.1)倍，SOX2上调(6.0±1.2)倍，NANOG上调(4.0±0.6)倍(P < 0.05；图1)。以上结果提示，实验已成功建立体外人喉鳞癌干细胞培养体系。采用Western Blot检测TRAP1在CD24-CD44+人喉鳞癌干细胞和CD24+CD44-人喉鳞癌细胞中的表达情况。结果显示，相比于非肿瘤干细胞，CD24-CD44+人喉鳞癌干细胞中TRAP1蛋白的表达明显上调(8.1±1.2)倍(P < 0.05；图2)。 2.2 siRNA抑制人喉鳞癌干细胞TRAP1表达 Western Blot实验结果显示，RNA干扰后，人喉鳞癌干细胞内TRAP1蛋白表达显著降低(图3)。 2.3 siRNA对人喉鳞癌干细胞周期的影响与对照组(未转染组)比较，TRAP1基因沉默后人喉鳞癌干细胞的生长速度明显减慢，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，见表1。 2.4 siRNA对人喉鳞癌干细胞增殖的影响 TRAP1基因沉默后人喉鳞癌干细胞的增殖受到明显的抑制，与对照组(未转染组)比较差异有显著性意义(P < 0.05)，见表2。 2.5 siRNA对人喉鳞癌干细胞克隆形成能力的影响相比于未经转染的人喉鳞癌干细胞，TRAP1基因沉默后人喉鳞癌干细胞克隆形成能力显著下调(图4A)。为进一步探究TRAP1对人喉鳞癌干细胞自我更新能力影响的具体机制，采用Western blot实验结果显示，相比于未经转染的人喉鳞癌干细胞，TRAP1基因沉默后人喉鳞癌干细胞内OCT4，SOX2，NANOG表达下调(图4B)。 2.6 siRNA对人喉鳞癌干细胞凋亡的影响 TUNEL法实验结果显示，相比于未经转染的人喉鳞癌干细胞，TRAP1基因沉默后人喉鳞癌干细胞更易于凋亡(图5A)。Western Blot实验结果显示，相比于未经转染的人喉鳞癌干细胞，TRAP1基因沉默后人喉鳞癌干细胞内凋亡基因BAD及BAX表达上调(图5B)。以上研究显示，TRAP1可促进人喉鳞癌干细胞凋亡。"

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