目的：探讨下调miR-221表达对子宫颈癌细胞恶性生物学行为的影响及其作用机制。方法：运用脂质体2000转染法将miR-221抑制剂和阴性对照核苷酸片段（NC）转染子宫颈癌细胞HeLa、C33A、SiHa和Caski细胞，用Western blotting和Real-time PCR分别检测ARID1A（miR-221靶蛋白）、cleaved caspase 3、caspase 3、cleaved PARP、PARP、MMP-9、MMP-13、TIMP-3蛋白表达水平和miR-221、 MMP-9、MMP-13、TIMP-3 mRNA的表达水平；用MTT法、克隆形成实验、AnnexinV-FITC/PI染色结合流式细胞术分别测定细胞增殖、克隆形成率和细胞凋亡率。结果：与阴性对照组比较,（1）miR-221抑制剂转染的HeLa、CC3A、SiHa和Caski细胞miR-221表达量均显著降低\[（0.23±0.01）、（0.31±0.02）、（0.34±0.01）、（0.37±0.02），F=25.44，P<005\]；（2）转染的细胞随着培养时间的增加细胞存活率逐渐下降，具有时间依赖效应，48 h之后细胞存活率显著低于阴性对照组（F=37.42，P<0.05）；细胞的克隆形成率显著降低（F=43.58，P<0.05）；培养72 h时细胞凋亡率分别为：HeLa（27.92±3.47）% 、C33A（20.84±4.31）%、SiHa（18.81±2.18）% 、Caski（19.86±3.82）%，均显著高于对照组（F=54.78，P<005）;（3）转染细胞下调miR-221表达后促进cleaved caspase 3、cleaved PARP、TIMP-3蛋白表达，抑制MMP-13蛋白表达； 降低MMP-13 mRNA表达（t=37.50，P<0.05），增加TIMP-3 mRNA表达（t=46.30，P<0.05)。结论：下调miR-221表达能抑制子宫颈癌细胞的恶性生物学行为，其机制与激活caspase信号通路从而诱导细胞凋亡以及调控MMP-13和TIMP-3的表达有关。

Objective:To explore effect of down-regulating miR-221 expression on malignant biologic behaviors of cervical cancer cells and its mechanism. Methods: Liposome 2000 transfection method was used to transfect miR-221 inhibitor and negative control nucleotide fragment (NC) into cervical cancer HeLa, C33A, SiHa and Caski line cells. Protein expression levels of ARIDIA (target protein of miR-221), cleaved caspase 3, caspase 3, cleaved PARP, PARP, MMP-9, MMP-13 and TIMP-3, as well as expression levels of miR-221, MMP-9, MMP-13, TIMP-3 mRNAs were respectively detected by Western blotting and Real-time PCR assays. Proliferation of the cells, clone formation rate and apoptosis rate of the cells were separately determined by MTT assay, clone formation experiment and flow cytometry assay plus AnnexinV-FITC/PI staining.Results: Comparing with the control group, (1) expression amounts of miR-221 in HeLa, CC3A, SiHa and Caski cells which transfected with miR-221 inhibitor were significantly decreased (\[0.23±0.01\], \[0.34±002\], \[0.34±0.01\] and \[0.37±0.02\], F=25.44, P<0.05); (2) survival rates of the transfected cells gradually decreased with increase of culture time, and had a time dependent effect, survival rates of the cells were significantly lower than that of the negative control group after culture for 48 h (F=37.42, P<0.05); clone formation rates of the cells obviously reduced (F=43.58, P<0.05); as culture for 72 h, apoptosis rates of the cells were (27.92±3.47)% for HeLa, (20.84±4.31)% for C33A, (18.81±2.18)% for SiHa and (19.86±3.82)% for Caski, obviously higher than that of the control group (F=54.78,P<0.05); (3) after expression of miR-221 in the transfected cells was down-regulated, expressions of cleaved caspase 3, cleaved PARP and TIMP-3 proteins were promoted, expression of MMP-13 protein was inhibited, expression of MMP-13 mRNA was depressed (t=37.5, P<0.05), and expression of TIMP-3 mRNA was increased (t=46.3, P<0.05). Conclusion: Down-regulation of miR-221 could inhibit malignant biologic behaviors of cervical cancer cells, its mechanism might be associated with activation of caspase signaling pathway which could induce apoptosis of the cancer cells and related with regulating and controlling expressions of MMP-13 and TIMP-3 proteins.

山东省医药卫生资助项目 (No. 2013WS0010)