miR-206抑制乳腺癌MDA-MB-231细胞Cdc42蛋白表达及其对细胞骨架的影响

miR-206 inhibits Cdc42 protein expression, invasion and migration of MDA-MB-231 cells

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 whether miR-206 can regulate Cdc42 protein expression and cytoskeleton remodeling. Methods miR-206 was transfected into MDA-MB-231 cells using LipofcctamineTM 2000, and the transfeeted cells were harvested in 48 h. Western blot analysis was used to detect the protein expressions of Cdc42, MMP-2 and MMP-9. Actin-Tracker Green was used to stain F-actin and filopodia was observed by fluorescent microscopy. Invasion assay and Transwell migration assay were taken to examine the invasive and migratory ability of ceils before and after the transfeetion respectively. Results Protein expressions of Cdc42, MMP-2 and MMP-9 were all down-regulated （P 〈 0.01 ）. The number of filopodia per cell in the blank group （ untransfected group） was 14.99 ±5.53, in EGF group was 23.59 ±3.92, miR-206 group was （9.45 ±3.59）, and miR-206 ＋ EGF group was 11.77 ±2.85. The filopodia numbers in miR-206 and miR206 ＋ EGF groups was much less than those in the blank group and EGF group （P 〈 0. 05）. The invasion and migration of miR-206 transfeeted MDA-MB-231 cells （65.0 ±13.9） were significantly lower than MDA-MB-231 cells [ blank control, 311.7 ±23.5, P 〈 0. 05 ] ; Transwell migratory assay indicated that there were more migrated cells in blank control group than in miR-206 group [ 793.0 ±76.3 vs 415.3 ±20.0, P 〈 0. 05 ]. Conclusion miR-206 downregulates Cdc42 protein expression in MDA-MB-231 cells, inhibits the formation of filopodia, and thus inhibits the invasion and migration efficiency of the cells.