沈纳　白广平　高天乐　黄新生　李俊

(1. 复旦大学附属中山医院耳鼻咽喉科，上海　200032；

2. 复旦大学附属中山医院青浦分院耳鼻咽喉科，上海　201700)

·论著·

上调miR-519d对喉鳞癌细胞增殖及凋亡的影响

沈纳1白广平2高天乐2黄新生1李俊2

(1. 复旦大学附属中山医院耳鼻咽喉科，上海200032；

2. 复旦大学附属中山医院青浦分院耳鼻咽喉科，上海201700)

摘要目的：研究miR-519d对喉鳞癌细胞增殖及凋亡的调控作用。方法： 采用RT-PCR、Western印迹等技术检测人喉癌上皮细胞株Hep-2及人支气管上皮细胞(human bronchial epithelium cell，HBE)中信号转导和转录激活子3(signal transducer and activator of transcription 3，STAT3)以及miR-519d的表达情况；通过质粒转染上调Hep-2细胞株中miR-519d的表达后，采用细胞增殖及凋亡检测技术观察转染细胞与对照细胞的增殖及凋亡情况。结果： STAT3 mRNA在Hep-2中的表达明显高于其在HBE细胞中的表达(P<0.05)，而miR-519d在Hep-2中的表达明显低于其在HBE中的表达(P<0.05)。STAT3在转染miR-519d后的Hep-2细胞中的表达明显下降(P<0.05)。转染后继续培养0～7 d，转染后Hep-2细胞的增殖率明显低于未转染细胞(P<0.05)。 转染后继续培养24 h，转染miR-519d的细胞凋亡率为(2.80±0.15)%，对照细胞的凋亡率为(0.92±0.09)% (P=0.000 4)。转染后继续培养72 h, 转染miR-519d的细胞凋亡率为(23.06±3.52)%，对照细胞的凋亡率为 (23.26±2.56)%(P=0.965 5)。结论： STAT3在Hep-2细胞中高表达，而相关的miR-519d呈低表达，通过上调miR-519d可以抑制STAT3的表达从而抑制肿瘤细胞的增殖，促进肿瘤细胞的凋亡。

关键词喉鳞癌；miRNA-519d；STAT3； 增殖；凋亡

中图分类号R739.65

文献标识码A

Effect of Up-Regulating miR-519d on Proliferation and Apoptosis of Laryngeal Squamous Cell CarcinomaSHENNa1BAIGuangping2GAOTianle2HUANGXinsheng1LIJun2

1.DepartmentofOtolaryngology,ZhongshanHospital,FudanUniversity,Shanghai200032,China; 2.DepartmentofOtolaryngology,QingpuBranchofZhongshanHospital,FudanUniversity,Shanghai201700,China

AbstractObjective： To investigate the effect of up-regulating miR-519d on proliferation and apoptosis of laryngeal squamous cell carcinoma. Methods： The expression of signal transducer and activator of transcription 3 (STAT3) and miR-519d in human laryngeal carcinoma epithelium cell line Hep-2 and human bronchial epithelium cell(HBE) were detected by real-time fluorescent quantitative polymerase chain reaction (RT-PCR) and Western blotting. After up-regulating miR-519d expression in Hep-2 cell line by plasmid transfection technique, the proliferation and apoptosis of transfected cells and control cells was detected by proliferation and apoptosis assays. Results： STAT3 mRNA expression in Hep-2 cells was significantly higher than that in HBE cells (P<0.05). However, miR-519d expression in Hep-2 cells was significantly lower than that in HBE cells (P<0.05). The STAT3 mRNA expression in the transfected Hep-2 cells decreased significantly. During 0 and 7 day of post-transfection culture, proliferation rate of transfected Hep-2 cells was significantly lower than that of untransfected cells (P<0.05). After 24 h culture, the apoptosis rate of miR-519d transfected cells was (2.8±0.15)%, while that of control cells was (0.92±0.09)% (P=0.000 4). After 72 h culture, the apoptosis rate of miR-519d transfected cells was (23.06±3.52)%, while that of control cells was (23.26±2.56)% (P=0.965 5). Conclusions： STAT3 shows high expression in Hep-2 cell, while the related miR-519d shows low expression. By up-regulating miR-519d, STAT3 expression could be suppressed, so as to suppress the proliferation of tumor cells and promote the apoptosis of tumor cells.

Key WordsLaryngeal squamous cell carcinoma;miRNA-519d;Signal transducer and activator of transcription 3;Proliferation;Apoptosis

喉癌是耳鼻咽喉科最常见的恶性肿瘤，占全身恶性肿瘤的5.7%～7.6%，近年来其发病率明显增长。喉癌中93%～99%为喉鳞状细胞癌。目前对喉癌特别是晚期喉癌尚无满意的治疗方法[1]。微小RNA(microRNAs, miRNAs)是基因表达的重要调控因子。研究[2]显示，许多miRNAs的功能与细胞增殖、分化和凋亡等有关；miRNAs直接或间接导致了肿瘤的发生，是肿瘤治疗研究的潜在靶点。我们在以往研究的基础上，采用生物信息学软件(TargetScan)对特异性miRNAs进行靶基因预测筛选，发现喉癌的多个特异性miRNAs有一个共同的靶基因—信号转导和转录激活子3(signal transducer and activator of transcription 3, STAT3)。通过调控STAT3，特异性的miRNAs可能在喉癌的发生发展中发挥重要作用。研究喉癌中特异性的miRNAs对STAT3的作用及其调控机制意义重大。……