过表达RhoD对人黑素瘤A375细胞骨架和迁移侵袭的影响

目的：探讨RhoD对人黑素瘤细胞株A375细胞骨架、迁移和侵袭能力等的影响及作用机制。方法实验分为A375?RhoD组和A375?增强型绿色荧光蛋白（EGFP）组，分别用携带RhoD的慢病毒载体和阴性对照慢病毒载体转染。罗丹明标记鬼笔环肽染色观察细胞骨架，Transwell小室实验检测细胞的迁移和侵袭能力，流式细胞仪检测细胞周期，Western印迹法检测丝切蛋白、磷酸化丝切蛋白、Diaph2和RhoD的表达。结果与A375?EGFP组细胞相比，A375?RhoD组细胞体积增大，应力纤维变细，软弱无力，黏着斑增多；丝状伪足形成增加；皱褶缘和板状伪足无明显变化。Transwell迁移实验显示，A375?EGFP组和A375?RhoD组平均每200倍视野下穿膜细胞数分别为152.67±11.23和72.67±5.03，两组间差异有统计学意义（t =11.25，P <0.05）。Transwell人工基底膜侵袭试验显示，A375?EGFP组和A375?RhoD组平均每200倍视野下穿膜细胞数分别为78.33±12.34和9.00±1，两组间差异有统计学意义（t=9.70，P<0.05）。A375?EGFP和A375?RhoD两组间G1期、S期和G2期细胞所占百分比差异均无统计学意义（P>0.05）。Western印迹结果显示，A375?RhoD组细胞可在RhoD的刺激下激活下游信号效应分子Diaph2，促进其表达，而A375?EGFP组未能激活，两组间丝切蛋白和磷酸化丝切蛋白表达差异不显著。结论 RhoD过表达可能通过下游效应分子Diaph2调节细胞骨架进而抑制A375细胞的迁移和侵袭能力。

Effects of RhoD overexpression on the cytoskeleton,migration and invasion of a human melanoma cell line A375

Objective To evaluate effects of RhoD overexpression on the cytoskeleton, migration and invasion of a human melanoma cell line A375, and to explore their mechanisms. Methods Cultured A375 cells were divided into 2 groups to be transfected with a lentiviral vector carrying the RhoD gene(A375?RhoD group)and a negative lentiviral vector expressing enhanced green fluorescent protein (EGFP)(A375?EGFP group) respectively. After additional culture for 24 hours, rhodamine?phalloidin was used to stain cytoskeletal proteins, Transwell assay was conducted to evaluate migratory and invasive activities of cells, flow cytometry to estimate cell cycle distribution, and Western?blot analysis to measure protein expressions of cofilin, phosphorylated cofilin(p?cofilin), Diaph2 and RhoD. Results Compared with the A375?EGFP group, the A375?RhoD group showed larger cells with thinner, softer and weaker stress fibers, more focal adhesions and filopodias, but no obvious changes in lamellipodias or ruffled borders. Transwell assay showed that the number of A375 cells crossing the polycarbonate membrane as well as that crossing the artificial basement membrane(matrigel)per high?power field(× 200)were significantly smaller in the A375?RhoD group than in the A375?EGFP group(migration assay: 72.67 ± 5.03 vs. 152.67 ± 11.23, t=11.25, P<0.05;invasion assay:9.00 ± 1.00 vs. 78.33 ± 12.34, t=9.70, P<0.05). There were no significant differences in proportions of cells in G1, S or G2 phase between the two groups (all P > 0.05). Western?blot analysis showed that RhoD overexpression activated and promoted the expression of the downstream signaling protein Diaph2 in the A375?RhoD group, which was not observed in the A375?EGFP group. In addition, there were no significant differences in expressions of cofilin or p?cofilin between the two groups. Conclusion Overexpression of RhoD may regulate the cytoskeleton and suppress migratory and invasive activities of A375 cells by activating the downstream effector molecule Diaph2.