Effects of specific interfering TACC3 gene expression on proliferation and apoptosis of CD133+CD44+ oral squamous cell carcinoma cells

doi:10.3969/j.issn.2095-4344.2017.25.010

Abstract

Abstract:

BACKGROUND: Studies have indicated that the abnormal expression of TACC3 is closely related to the occurrence and development of many kinds of tumors, and the expression of TACC3 is up-regulated in these tumors. Therefore, in vitro specific inhibition of TACC3 expression may become an important target for the treatment or intervention of tumor growth.
OBJECTIVE: To investigate the mechanism by which TACC3 gene expression regulates cell proliferation and apoptosis in oral squamous cell carcinoma.
METHODS: CD133⁺CD44⁺ oral squamous cell carcinoma cells were sorted from human oral squamous cell carcinoma cell line Cal-27 by immunomagnetic beads. In experimental group, the shRNA sequence of TACC3 was designed and synthesized, which was then trasnfected into CD133⁺CD44⁺ oral cancer stem cells by Lipofectamine^TM 2000. Empty vector-trasnfected (negative control) and untransfected cells were used as callsed. Forty-eight hours after the transfection, effects of TACC3 gene silencing on proliferation and apoptosis in vitro in CD133⁺CD44⁺ oral squamous cell carcinoma were detected by MTT, clone formation test, and TUNEL assay. Western blot assay was used to detect the effect of TACC3 gene silencing on Ki67, Bax and Bcl-2 protein expression in CD133⁺CD44⁺ oral squamous cell carcinoma.
RESULTS AND CONCLUSION: (1) Cell proliferation. The proliferation rate and expression level of Ki67 were significantly lower in the experimental group than the negative control and untransfected groups (P < 0.05). (2) Clone formation. The clone formation ability in the experimental group was significantly lower than that in the negative control and untransfected groups (P < 0.05). (3) Cell apoptosis. TACC3 gene silencing caused an obvious decrease in Bcl-2 protein expression and a significant increase in Bax protein expression. These findings further confirmed that specific interference of TACC3 gene expression could inhibit the proliferation of CD133⁺CD44⁺ cells and promote the apoptosis.

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: Neoplastic Stem Cells, Mouth Neoplasms, Cell Proliferation

CLC Number:

R394.2

Duan Rui, Li Yong-sheng, Xia Yi-chao. Effects of specific interfering TACC3 gene expression on proliferation and apoptosis of CD133⁺CD44⁺ oral squamous cell carcinoma cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(25): 3995-4000.

Figures/Tables 1

2.1 口腔鳞癌干细胞的分离与鉴定利用免疫磁珠分选技术，从人口腔鳞癌细胞系Cal-27中分选获得CD133+ CD44+和CD133-CD44-两种细胞。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(SOX2、NANOG、OCT4)的表达。结果显示，相比于CD133-CD44-的人口腔鳞癌细胞，CD133+CD44+口腔鳞癌细胞内SOX2、NANOG、OCT4均呈现高表达，见图1。以上结果提示CD133+CD44+双阳性细胞为口腔鳞癌干细胞。采用Western blot检测TACC3在CD133+CD44+和CD133-CD44-口腔鳞癌细胞中的表达情况。结果显示，相比于CD133-CD44-口腔鳞癌细胞，CD133+CD44+口腔鳞癌干细胞中TACC3的表达明显上调(0.55±0.16，0.26±0.20，P < 0.05)，见图2。 2.2 shRNA抑制口腔鳞癌干细胞TACC3表达 Western blot实验结果显示，RNA干扰后，CD133+CD44+口腔鳞癌干细胞内TACC3蛋白表达显著降低(图3)。 2.3 shRNA干扰抑制TACC3表达对CD133+CD44+口腔鳞癌干细胞增殖的影响 MTT检测结果发现，shRNA转染组TACC3基因沉默后CD133+CD44+口腔鳞癌干细胞的增殖速度明显低于未转染组和阴性对照组(图4A)。此外，采用Western blot检测增殖相关蛋白Ki67在细胞中的表达水平，发现shRNA转染组细胞中Ki67蛋白表达水平明显低于未转染组和阴性对照组(图4B)。 2.4 shRNA干扰抑制TACC3表达对CD133+CD44+口腔鳞癌干细胞克隆形成能力的影响相比于未转染组和阴性对照组，shRNA转染组CD133+CD44+口腔鳞癌干细胞克隆形成能力显著下调(图5)。 2.5 shRNA干扰抑制TACC3表达对CD133+CD44+口腔鳞癌干细胞凋亡的影响相比于未转染组和阴性对照组，shRNA转染组更易于凋亡(图6A)。Western blot实验结果显示，shRNA转染组口腔鳞癌干细胞促凋亡蛋白Bax表达水平显著高于未转染组和阴性对照组，而抗凋亡蛋白Bcl-2表达则相反(图6B)。"

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