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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、Annexin V-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He La、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:He La(27.92±3.47)%、C33A(20.84±4.31)%、Si Ha(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 investigate the effect of miR-221 expression on the malignant biological behavior of cervical cancer cells and its mechanism. Methods: The cervical cancer cell lines HeLa, C33A, SiHa and Caski were transfected with miR-221 inhibitor and negative control nucleotide fragment (NC) by lipofectamine 2000. Western blotting and Real-time PCR The expressions of ARID1A, cleaved caspase 3, caspase 3, cleaved PARP, PARP, MMP-9, MMP-13 and TIMP- 3 mRNA were detected by MTT assay, colony formation assay and flow cytometry. Annexin V-FITC / PI staining and flow cytometry were used to detect the cell proliferation, colony formation and apoptosis rate respectively. RESULTS: Compared with the negative control group, (1) miR-221 expression in He La, CC3A, SiHa and Caski cells transfected with miR-221 was significantly lower than that in the negative control group [(0.23 ± 0.01), 0.34 ± 0.01), (0.37 ± 0.02), F = 25.44, P <0.05]. (2) The cell survival rate of transfected cells decreased gradually with the increase of culture time and had a time-dependent effect. (F = 43.58, P <0.05). The apoptotic rates at 72 h after culture were He La (27.92 ± 3.47, P <0.05) ), C33A (20.84 ± 4.31)%, Si Ha (18.81 ± 2.18)% and Caski (19.86 ± 3.82)%, respectively, which were significantly higher than that of the control group (F = 54.78, After down-regulating the expression of miR-221, the expression of cleaved caspase 3, cleaved PARP and TIMP-3 protein was down-regulated and the expression of MMP-13 protein was downregulated (P <0.05) t = 46.30, P <0.05). Conclusion: The down-regulation of miR-221 expression can inhibit the malignant biological behavior of cervical cancer cells. The mechanism is related to the activation of caspase signaling pathway to induce apoptosis and the regulation of MMP-13 and TIMP-3 expression.