Abstract:

Wen Sijian, Ni Nana, Zhou Xiaowei, Wu Qiong, Wang Xiaopo, Song Hao, Zhang Wei, Sun Jianfang Department of Pathology, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China （Wen SJ, Wu Q, Wang XP, Song H, Zhang W, Sun JF）; Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China （Ni NN, Zhou XW） Corresponding author: Sun Jianfang, Email: fangmin5758@aliyun.com 【Abstract】 Objective To evaluate effects of RhoD over 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 s 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 over activated and promoted the 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 s of cofilin or p?cofilin between the two groups. Conclusion Over of RhoD may regulate the cytoskeleton and suppress migratory and invasive activities of A375 cells by activating the downstream effector molecule Diaph2.