天津医药

天津医药 ›› 2016, Vol. 44 ›› Issue (10): 1185-1189.doi: 10.11958/20150403

• 细胞与分子生物学 •    下一篇

长链非编码 RNA HOTAIR 影响人舌鳞癌细胞 Tb3.1 增殖与凋亡的体内外研究

郭文宇,孔令平,孙珊珊,王宇,赵明慧,周旋,王旭东,张仑   

  1. 天津医科大学肿瘤医院颌面耳鼻喉科， 国家肿瘤临床医学研究中心， 天津市肿瘤防治重点实验室（邮编 300060）
  • 收稿日期:2015-12-16 修回日期:2016-07-19 出版日期:2016-10-15 发布日期:2016-10-21
  • 通讯作者: 张仑 E-mail:327330653@qq.com
  • 作者简介:郭文宇（1990）， 男， 硕士在读， 主要从事头颈部肿瘤的诊治研究
  • 基金资助:
    国家自然科学基金资助项目（81172573）

Effects of long non-coding RNA HOTAIR on proliferation and apoptosis of human tongue squamous cell carcinoma in vitro and in vivo

GUO Wenyu, KONG Lingping, SUN Shanshan, WANG Yu, ZHAO Minghui, ZHOU Xuan, WANG Xudong, ZHANG Lun   

  1. Department of Maxillofacial & E.N.T（ear, nose, and throat）Oncology, Tianjin Medical University Cancer Institute and Hospital; National Clinical Research Center of Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, China
  • Received:2015-12-16 Revised:2016-07-19 Published:2016-10-15 Online:2016-10-21
  • Contact: ZHANG Lun E-mail:327330653@qq.com

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摘要： 目的 研究长链非编码 RNA HOTAIR 对人舌鳞癌细胞系 Tb3.1 增殖与凋亡的影响 。 方法 使用 HOTAIR siRNA（siHOTAIR）敲低 HOTAIR 的表达； 实验分为 siHOTAIR 组、无义序列组和空白对照组。 前 2 组分别用 siHOTAIR 和无义序列转染舌鳞癌细胞， 空白对照组细胞不做任何处理。 实时定量 PCR 检测 HOTAIR 的表达水平； 四甲基偶氮唑蓝（MTT）法检测 Tb3.1 细胞增殖； Western blot 检测 B 细胞淋巴瘤 2（Bcl-2）、活化型半胱氨酸天冬氨酸蛋白酶-3（Cleaved Caspase-3）、BAX 的表达； 流式细胞仪检测细胞凋亡； 平板克隆形成实验检测细胞增殖情况；并行细胞衰老检测 。 动物实验建立 Tb3.1 裸鼠皮下荷瘤模型， 通过免疫组织化学染色及 TUNEL 法评价干扰 HOTAIR 后对 Tb3.1 细胞增殖、凋亡的作用。 结果 siHOTAIR 处理后 HOTAIR 的表达水平降低； Western blot 结果示 Cleaved Caspase-3 和 BAX 表达水平升高， Bcl-2 表达水平降低。 MTT 结果显示 siHOTAIR 组细胞增殖受到抑制；流式细胞示 siHOTAIR 组细胞凋亡升高； 细胞衰老实验显示 siHOTAIR 组细胞衰老数目增加； 免疫组化结果显示， siHOTAIR 组较对照组 Ki-67、Bcl-2 表达减少， Caspase-3、BAX 表达增加； TUNEL 结果显示， siHOTAIR 组较空白对照组肿瘤细胞凋亡水平升高。 结论 干扰 HOTAIR 可以促进舌鳞癌细胞的凋亡并抑制其增殖， HOTAIR 可以作为舌鳞癌治疗的研究方向。

关键词: 舌肿瘤, 癌, 鳞状细胞, RNA, 小分子干扰\细胞增殖, 细胞凋亡, HOTAIR

Abstract: Objective To investigate the influence of long non-coding RNA HOTAIR in proliferation and apoptosis of human tongue squamous cell carcinoma in vitro and in vivo. Methods siHOTAIR was used to inhibit the HOTAIR expression in Tb3.1 human tongue squamous cell carcinoma cell line. The experiments were divided into siHOTAIR group, nonsense sequence group and blank control group. Real-time PCR was used to detect the HOTAIR expression. MTT assay was employed to determine the cell survival. The expression levels of Bcl2, BAX, caspase- 3, cleaved caspase- 3 were examined by Western blot assay. Tb3.1 xenograft tumor model was established in BALB/c nude mice, and the tumor model was divided into control group, negative group, and siHOTAIR treated group. The tumor tissues were measured by immunohistochemistry stain (IHC) and TUNEL assay. Results The detection of real- time PCR showed that HOTAIR expression was reduced after treated with siHOTAIR. Western blots assay showed that Bcl-2 protein was suppressed while cleaved caspase- 3 and BAX protein were up-regulated after treated with siHOTAIR. MTT assay indicated that the cell survival rate was significantly reduced in siHOTAIR treated group. Flow cytometry detected that apoptosis levels were increased in siHOTAIR group. The level of cell senescence was higher in the siHOTAIR group than that of control group. Results of IHC indicated that Ki-67 and Bcl-2 protein of tumor tissue were inhibited, while BAX and cleaved caspase- 3 protein expressions were elevated simultaneously in the siHOTAIR group. TUNEL assay suggested that more apoptosis was observed in siHOTAIR group. Conclusion HOTAIR can affect proliferation and apoptosis of tongue squamous cancer cells. HOTAIR may be one of the new candidate targets for human tongue cancer therapy.

Key words: tongue neoplasms, carcinoma, squamous cell, RNA, small interfering, cell proliferation, apoptosis, HOTAIR