Wang Xiao-mei, Xu Jing, Cheng Zhi-qiang, Peng Quan-zhou, Hu Jin-tao, Gao Li-kun, Zhang Shi-fen, Jin Hong-tao
Department of Pathology, the Second Affiliated Hospital, Jinan University/Shenzhen People's Hospital, Shenzhen 518020, China.
Zhonghua Bing Li Xue Za Zhi. 2012 Apr;41(4):254-9. doi: 10.3760/cma.j.issn.0529-5807.2012.04.009.
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.
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.
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 antisense 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).
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.
探讨微小RNA-21（miR-21）反义寡核苷酸对人宫颈鳞癌细胞系SiHa体内外生物学特性的影响。
合成针对miR-21的特异性硫代磷酸反义寡脱氧核苷酸，并体外转染至宫颈癌细胞。采用实时RT-PCR检测转染后SiHa细胞中miR-21的表达。通过MTT法和集落形成实验评估细胞增殖。采用膜联蛋白V-FITC/PI分析检测细胞凋亡。通过肿瘤生长曲线和免疫组织化学（MaxVision法）评估miR-21反义寡核苷酸对肿瘤生长的抑制作用。采用苏木精-伊红（H-E）染色记录形态学变化，采用荧光TUNEL法检测凋亡活性。
与对照组相比，转染反义miR-21后，miR-21表达下降，同时细胞生长明显受到抑制（P<0.05）。与阴性组相比，反义miR-21显著抑制了两种细胞系的集落形成（55.6%±1.4%）（阴性组为98.3%±2.0%，P<0.05）。流式细胞术检测显示，反义miR-21表达显著增强细胞凋亡（6.7%±1.3%和29.4%±1.7%，P<0.05）。miR-21转染组和阴性对照组的成瘤率分别为3/8和6/8（P<0.05）。与阴性对照组相比，转染组中Ki-67增殖标志物染色显著降低（42%对90%）。在miR-21转染细胞中可见大量死亡肿瘤细胞，同时凋亡明显增加（P<0.05）。
靶向反义寡核苷酸miR-21通过诱导凋亡有效抑制宫颈癌细胞系SiHa的体内外生长。
