目的

探讨微小RNA-21（miR-21）反义寡核苷酸（miR-21 ASO）对舌鳞癌细胞增殖和凋亡的调控作用。

方法

通过转染miR-21 ASO，采用实时荧光定量RT-PCR（qRTPCR）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-21 ASO 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-21 ASO 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-21 ASO 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-21 ASO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-21 ASO 对舌鳞癌细胞miR-21 水平降低具有靶向特异性，转染miR-21 ASO 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 a nalyzed 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）.MTT 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.