摘要目的 探讨体内外微小RNA-21( miR-21)反义寡核苷酸对宫颈鳞癌SiHa细胞株绌胞生物学特性的影响.方法 miR-21反义寡核苷酸(miR-21-ASO)通过脂质体转染宫颈鳞癌SiHa细胞,即时定量逆转录聚合酶链反应(RT-PCR)检测miR -21表达情况,采用四甲基偶氮唑盐(MTT)比色法、集落形成实验、流式细胞术等方法探讨抑制miR-21表达对SiHa细胞形态及生物学特性改变的影响.应用免疫组织化学MaxVision法、TUNEL( TdT-mediated dUTP nick end labeling)细胞凋亡检测试剂盒检测移植瘤细胞增殖活性和凋亡情况.结果 miR-21在SiHa细胞株中呈显著高表达,miR-21-ASO可使SiHa细胞中miR-21表达量显著降低(P＜0.05).MTT法检测结果显示,转染miR-21 -ASO后SiHa细胞的生长受到明显抑制(P＜0.05).阴性对照组的细胞集落形成率为98.3％±2.0％,miR-21抑制组则为55.6％±1.4％,miR-21抑制组的细胞集落形成率受到明显抑制(P＜0 05).流式细胞术检测显示,阴性对照组和miR-21抑制组的SiHa细胞凋亡率分别为6.7％±1.3％和29.4％±1.7％,miR-21抑制组的细胞凋亡率明显增加(P＜0.05).裸鼠皮下移植瘤成瘤率,miR-21抑制组与阴性对照组的成瘤比例分别为3/8和6/8(P ＜0.05).裸鼠瘤体的Ki-67阳性指数,miR-21抑制组(42％)比阴性对照组(90％)显著下降.荧光TUNEL凋亡检测显示,miR-21抑制组裸鼠肿瘤细胞凋亡明显增多(P＜0.05).结论 miR-21-ASO可有效抑制宫颈鳞癌SiHa细胞中miR-21表达,影响癌细胞增殖活性,促进癌细胞凋亡,体内抑制SiHa细胞裸鼠移植瘤生长促进其凋亡.

abstractsObjective To explore the effect of microRNA-21 (miR-21) antisense oligonucleotide on the biological characteristics of human cervical squamous carcinoma cell lines SiHa in vivo and in vitro.Methods Specific phosphorothioate antisense oligodeoxynucleotides targeting miR-21 were synthesized and transfected into cervical cancer cells in vitro.Expression of miR-21 in SiHa after transfection was detected by real-time RT-PCR.The cell proliferation was evaluated by MTT assay and colony formation experiment.The cell apoptosis was analyzed by annexin V-FITC/PI analysis. The inhibitory effect of miR-21 antisense oligonucleotide on tumor growth was evaluated by tumor growth curves and immunohistochemistry ( MaxVision method).H-E staining was used to document morphological changes and fluorometric TUNEL assay was to detect the apoptotic activity.Results After the transfection of antisense miR-21,the expression of miR-21 decreased along with an obvious growth inhibition,compared with that of the control groups ( P ＜ 0.05).Colony formation of both cell lines was markedly inhibited with antisense miR-21 ( 55.6％ ± 1.4％ ),as compared with that in the negative group (98.3％ ±2.0％,P ＜0.05).Flow cytometry assay showed that antiscnse miR-21 expression significantly enhanced the cell apoptosis (6.7％ ± 1.3％ and 29.4％ ± 1.7％,P ＜0.05 ).The tumor-forming rates of miR-21 transfected group,and negative control groups were 3/8 and 6/8,respectively (P ＜0.05).Ki-67 proliferative marker staining decreased significantly (42％ vs 90％ ) in the transfected group compared with negative control groups.Extensive dead tumor cells were seen in the miR-21 transfected cells along with a marked increase of apoptosis (P ＜ 0.05 ). Conclusion Targeted antisense oligonucleotide miR-21 effectively suppresses the growth of cervical carcinoma SiHa cells both in vitro and in vivo through an induction of apoptosis.