microRNA―34体外调控鼻咽癌鳞状肿瘤干细胞表型.docVIP

microRNA―34a体外调控鼻咽癌鳞状肿瘤干细胞表型 　　[摘要] 目的 探讨microRNA-34a（miR-34a）对鼻咽癌鳞状肿瘤干细胞表型的体外调控作用。 方法 利用原代培养的人类鼻咽癌细胞，采用流式分选法定量并收集ALDH（aldehyde dehydrogenase）+肿瘤干细胞。通过RT-qPCR分析miR-34与肿瘤干细胞相关因子（Sox2、Nanog、Oct3/4）在ALDH+和ALDH-细胞中的表达情况。进一步用miR-34a拟态转染评估其对鼻咽癌肿瘤干细胞标记物以及相关因子的表达调控能力。 结果 原代鼻咽癌细胞中表达ALDH的细胞约占15.43%左右。与ALDH-细胞相比，miR-34a表达水平在大多数ALDH+细胞中显著下调（-13.2 vs -4.1倍），3种肿瘤干细胞相关因子表达显著增加（最高36.8倍）。ALDH+细胞转染miR-34a拟态24 h、48 h、72 h后miR-34a mRNA水平显著增加，48 h时达峰值水平（4.49倍，P中国论文网 /6/viewhtm　　[关键词] 鼻咽癌；肿瘤干细胞；microRNA-34a；转染；拟态；醛脱氢酶 　　[中图分类号] R76 [文献标识码] A [文章编号] 1673-9701（2016）21-0028-05 　　[Abstract] Objective To investigate the role of miR-34a in regulating cancer stem cell（CSC） phenotype of nasopharyngeal squamous cell carcinoma in vitro. Methods The cells were isolated from patients with nasopharyngeal carcinoma （NPC） and cultured in vitro. Flow-cytometry was used to quantify and enrich for ALDH+ cancer stem cells （CSCs）. RT-qPCR was performed to analyze expression patterns of miR-34a and CSC-related transcription factors（CSC-TFs） （Sox2， Nanog， Oct3/4） for ALDH+ and ALDH-cells. Transfection of miR-34a mimics was used to evaluate its regulatory potential for CSC marker profiles as well as CSC-TFs expression in NPC-CSC. Results The expression of ALDH was found in around 15.43% of primary NPC cells. miR-34a expression levels were significantly downregulated in the majority of ALDH+ cells derived from primary NPC as compared to ALDH-cells （-13.2 vs -4.1 fold）. For CSC-related TF expression， ALDH+ cells showed a significantly increased level compared to ALDH-cells（up to 36.8 fold）. miR-34a mRNA level in ALDH+ cells after transfection with miR-34a mimics significantly increased after 24 h， 48 h and 72 h， and the peak of miR-34a level was found 48 h post-transfection（4.49 fold， P 　　1 材料与方法 　　1.1 鼻咽癌原代细胞培养 　　新鲜鼻咽鳞状细胞癌组织由本院耳鼻喉科提供。本研究经患者知情同意和医院伦理委员会审查通过，研究工作从2015年8月开展至今。步骤：①适量新鲜标本置于15 mL离心管中，装2 mL含10?GFBS（Gibco公司）的RPMI-1640（Gibco公司）培养基。②去除外周坏死组织，无血清培养基充分漂洗3次。③将组织块剪成0.5 mm大小，加入适量0.25?G胰酶，37℃消化8～10