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目的探讨miR-206对MDA-MB-231乳腺癌细胞Cdc42表达的调控及对细胞骨架的影响。方法采用Li-pofectamineTM2000转染miR-206进入MDA-MB-231细胞,48h后Western blot检测转染后乳腺癌细胞Cdc42、MMP-2和MMP-9蛋白质表达。用免疫荧光显微镜观察细胞丝状伪足的改变,进一步用侵袭迁移实验检测转染前后细胞侵袭力和迁移力的变化。结果Western blot检测Cdc42、MMP-2和MMP-9在转染前后的表达结果,其条带灰度值与阴性对照组相比明显下降(P<0.05);细胞免疫荧光计数每个细胞平均丝状伪足数,空白对照组为(14.99±5.53),表皮生长因子(EGF)组为(23.59±3.92),miR-206转染组为(9.45±3.59),miR-206+EGF组为(11.77±2.85)。与空白对照组和EGF组比较,miR-206转染组或miR-206+EGF组细胞丝状伪足明显减少(P<0.05)。侵袭实验结果显示,小室膜下的细胞数量空白对照组为(311.7±23.5),miR-206转染组为(65.0±13.9),二者差异有统计学意义(P<0.01)。迁移实验结果显示,小室膜下的细胞数量空白对照组为(793.0±76.3),而miR-206转染组为(415.3±20.0),二者差异有统计学意义(P<0.01)。结论miR-206能抑制MDA-MB-231细胞Cdc42的表达和细胞丝状伪足的形成,并且能抑制细胞的侵袭迁移能力。
Objective To investigate the regulation of miR-206 on the expression of Cdc42 in MDA-MB-231 breast cancer cells and its effect on cytoskeleton. Methods The miR-206 was transfected into MDA-MB-231 cells by Li-pofectamineTM2000, and the protein expression of Cdc42, MMP-2 and MMP-9 was detected by Western blot 48 h after transfection. Immunofluorescence microscopy was used to observe the change of filamentous pseudopods. The invasion and migration of cells were detected by invasion and migration assay. Results The expression of Cdc42, MMP-2 and MMP-9 before and after transfection were detected by Western blot. The gray values of the bands were significantly decreased compared with those of the negative control group (P <0.05). The immunofluorescence counts of cells per cell (14.99 ± 5.53) in the blank control group, (23.59 ± 3.92) in the EGF group and (9.45 ± 3.59) in the miR-206 transfected group, and 11.77 ± 2.85). Compared with the blank control group and the EGF group, the filopodia of miR-206 transfection group or miR-206 + EGF group were significantly decreased (P <0.05). The results of invasion assay showed that the number of cells in the ventricular membrane was (311.7 ± 23.5) in the blank control group and (65.0 ± 13.9) in the miR-206 transfected group, the difference was statistically significant (P <0.01). Migration assay showed that the number of cells in the ventricular membrane was (793.0 ± 76.3) in blank control group and (415.3 ± 20.0) in miR-206 transfected group, the difference was statistically significant (P <0.01). Conclusion miR-206 can inhibit the expression of Cdc42 and the filopodia of MDA-MB-231 cells and inhibit the invasion and migration of cells.