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). 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. Key words: Melanoma, experimental; GTP phosphohydrolases; Cytoskeleton; Cell migration assays; A375 cells; RhoD