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目的:探讨miRNA-200c的表达对卵巢癌细胞侵袭能力的影响,并初步分析其可能的作用机制。方法:应用Lipofectamine 2000瞬时上调ES-2细胞中miR-200c的表达,并通过荧光定量PCR验证;Transwll侵袭小室检测细胞侵袭能力的变化;生物信息学的方法预测miR-200c的靶基因,同时荧光素酶报告基因实验验证E盒结合锌指蛋白2(zinc finger E-box bingding homeobox 2,ZEB2)是miR-200c的靶基因;蛋白印迹法检测E-钙黏蛋白和波形蛋白的表达水平。结果:pre-miR-200c转染ES-2细胞后,转染组miR-200c的表达(4.82±1.73)较对照组(1.00±0.16)明显升高,t=-67.28,P<0.001;上调miR-200c后,转染组(27.70±1.19)与对照组(57.30±2.01)相比,细胞的侵袭能力明显减弱,t=11.17,P<0.001;蛋白印迹法的结果显示,转染组ZEB2的表达水平为0.38±0.015,较对照组的0.66±0.024明显下降,t=94.67,P<0.001。采用生物信息学的方法预测ZEB2是miR-200c的靶基因;且荧光素酶报告实验结果证实,共转染野生型荧光素酶质粒和pre-miR-200c,与其他各组相比,荧光素酶活性明显降低,差异有统计学意义,P<0.001;转染pre-miR-200c后,转染组E-钙黏蛋白的表达(0.75±0.023)较对照组(0.32±0.014)明显增加,t=-97.77,P<0.001,波形蛋白的表达为0.28±0.010,较对照组0.67±0.129明显降低,t=71.64,P<0.001。结论:miR-200c能通过靶向ZEB2抑制人卵巢癌细胞的侵袭能力,其异常表达与卵巢癌细胞的生物学行为密切相关。
Objective: To investigate the effect of miRNA-200c on the invasiveness of ovarian cancer cells and to analyze its possible mechanism. METHODS: The expression of miR-200c in ES-2 cells was transiently up-regulated by Lipofectamine 2000 and verified by real-time PCR. Transwll invasion chamber was used to detect the invasion of cells. Bioinformatics method was used to predict the miR-200c target gene, The enzyme reporter gene was used to verify that zinc finger E-box binding homeobox 2 (ZEB2) is the target gene of miR-200c. The expression of E-cadherin and vimentin were detected by Western blotting. Results: After transfection with pre-miR-200c, the expression of miR-200c (4.82 ± 1.73) was significantly higher than that of the control group (1.00 ± 0.16), t = -67.28, P <0.001; Compared with the control group (57.30 ± 2.01), the invasion ability of miR-200c transfected group (27.70 ± 1.19) was significantly weaker (t = 11.17, P <0.001). The result of Western blotting showed that the transfection group ZEB2 The expression level was 0.38 ± 0.015, which was significantly lower than that of the control group (0.66 ± 0.024, t = 94.67, P <0.001). Bioinformatics method was used to predict that ZEB2 was the target gene of miR-200c; and luciferase reporter assay confirmed that transfected wild-type luciferase plasmid and pre-miR-200c, compared with other groups, (P <0.001). The expression of E-cadherin (0.75 ± 0.023) in transfection group was significantly increased compared with that in control group (0.32 ± 0.014) after transfected with pre-miR-200c, the difference was statistically significant t = -97.77, P <0.001, the expression of vimentin was 0.28 ± 0.010, which was significantly lower than that of the control group (0.67 ± 0.129, t = 71.64, P <0.001). Conclusion: miR-200c can inhibit the invasion of human ovarian cancer cells by targeting ZEB2. The abnormal expression of miR-200c is closely related to the biological behavior of ovarian cancer cells.