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

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。

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

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.