微小RNA-21反义寡核苷酸对舌鳞癌细胞增殖和凋亡的调控作用  

Regulation of microRNA-21 ASO on proliferation and apoptosis of TSCC cell lines

作　　者：李劲松[1] 黄洪章[2] 潘朝斌[1] 陈伟良[1] 武东辉[1] 林钊宇[1] 张佩琢 

机构地区：[1]中山大学附属第二医院口腔颌面外科,广州510120 [2]中山大学光华口腔医学院.附属口腔医院.口腔医学研究所 [3]苏州吉玛基因药物科技有限公司

出　　处：《中华口腔医学研究杂志（电子版）》2009年第2期17-22,共6页Chinese Journal of Stomatological Research(Electronic Edition)

基　　金：广东省自然科学基金项目(7001593);广东省科技计划项目(2008B030301132);广州市科技计划项目(2008Z1-E201);国家自然科学基金(30672333)

摘　　要：目的探讨微小RNA-21(miR-21)反义寡核苷酸(miR-21ASO)对舌鳞癌细胞增殖和凋亡的调控作用。方法通过转染miR-21ASO,采用实时荧光定量RT-PCR(qRT-PCR)检测舌鳞癌细胞株SCC-15和CAL27中miR-21的表达变化,荧光素酶活性检测实验分析miR-21ASO在舌鳞癌细胞中的调控功能,并运用多种细胞增殖和凋亡检测方法以观察miR-21ASO对舌鳞癌细胞调控产生的系列效应。结果miR-21在两株舌鳞癌细胞SCC-15和CAL27中的表达显著高于在正常舌鳞状上皮细胞中的表达(P=0.037,0.015),转染miR-21ASO可显著抑制其表达(P=0.017,0.006),miR-21对荧光素酶活性的抑制率由50%显著减少到20%以下(P=0.002,0.003),SCC-15和CAL27细胞存活率比转染前明显降低(P=0.002,0.004),细胞克隆形成率比转染前明显降低(P=0.007,0.032);流式细胞仪检测显示转染miR-21ASO后,SCC-15和CAL27细胞凋亡明显增加,激光共聚焦显微镜观察到线粒体细胞色素C释放较转染前增加。结论miR-21ASO对舌鳞癌细胞miR-21水平降低具有靶向特异性,转染miR-21ASO可有效抑制miR-21的促癌效应,利用反义核酸技术的高度特异性开展针对促癌microRNA分子的靶向治疗将可能为舌鳞癌的基因治疗开辟新的途径。Objective To study the regulation effect of miR-21 ASO on proliferation and apoptosis of Tongue squamous cell carcinoma （TSCC） cell lines. Methods TSCC cell lines： SCC-15 and CAL27 were cultured in vitro and their miR-21 expressions change were determined by transfection of miR-21 ASO. Luciferase reporter assay was used to evaluate the function of miR-21 ASO. The cell proliferation following transfection was evaluated by MTT assay and colony formation experiment. The cell apoptosis following transfection was analyzed by Annexin V analysis and Cytochrome C release experiment. Results MiR-21 was up-regulated highly 14-fold in SCC-15 and CAL27 cell lines relative to the normal tongue squamous cell （NTSC）（P=0.037,0.015）, but after transfected with miR-21 ASO, miR-21 expression in the cell lines was knocked down significantly （P=0.017,0.006）, meanwhile the inhibition of luciferase activity by miR-21 reduced from 50% to 20% （P=0.002,0.003）. MTF assay revealed that viability of SCC-15 cells reduced from 86% to 45% （P=0.002） after transfection with miR-21 ASO, while that of CAL27 cells decreased from 83% to 39% （P=0.004）. Colony formation of both cell lines decreased highly after transfected with miR-21 ASO （P=0.007,0.032）. Annexin V assay showed that transfection with miR-21 ASO promoted apoptosis greatly. The percent of apoptotic cells increased 27 folds for SCC-15 cells and 17 folds for CAL27 cells. Similarly, miR-21 ASO transfected cells displayed Cytochrom C release. Conclusions MiR-21 function can be antagonized effectively by miR-21 ASO. The specificity and effectiveness of miR-21 ASO in antagonizing miR-21 function hold great promise for the development of therapeutic strategies based on miR-21 inhibition.

关 键 词：舌鳞癌 MIR-21 反义寡核苷酸 增殖 凋亡 

分 类 号：R739.8[医药卫生—肿瘤]