目的:观察Wiskoot-Aldrich综合征蛋白家族成员3(WASF3)对乳腺癌细胞增殖、迁移侵袭及伪足形成的影响。方法:利用免疫蛋白印迹(Western blot)法检测乳腺癌细胞(BT-549、HCC-1937、BT-474、MDA-MB-231、MCF-7、T-47D)及乳腺正常细胞(MCF-10A)中WASF3蛋白的表达量；获取WASF3互补脱氧核糖核苷酸(cDNA)，构建过表达及干扰的重组载体SB-16-WASF3和pHB-U6-MCS-PGK-PURO-shWASF3(pHB-shWASF3)。重组干扰质粒经慢病毒包装，感染MDA-MB-231细胞，经抗性筛选建立WASF3基因静默的稳转细胞株。经实时荧光定量反转录聚合酶链反应(RT-qPCR)及Western blot法验证WASF3的静默及过表达效率；通过克隆形成实验和迁移侵袭实验分别验证细胞克隆形成能力和迁移侵袭能力的变化；过表达载体SB-16-WASF3转染MDA-MB-231细胞，经WASF3抗体和鬼笔环肽(Phalloidin)双染色，观察WASF3过表达对细胞伪足形成的影响。采用单因素方差分析，组内进一步两两比较采用LSD-n t检验，两两比较采用n t检验。n 结果:Western blot结果显示，不同乳腺癌细胞WASF3蛋白的相对表达量分别为1.07±0.00、0.54±0.11、0.60±0.14、1.36±0.02、0.65±0.05和0.79±0.18，均高于乳腺正常细胞(0.38±0.01，n t＝134.864、21.289、24.883、55.397、8.892、37.363，n P值均<0.05)，差异均有统计学意义；且MDA-MB-231和BT-549细胞的表达量相对较高。细胞增殖实验显示WASF3静默组(pHB-shWASF3-1)在24、48、72、96、120 h的增值率均低于对照组pHB-shNegetive Control组(pHB-shNC)及空白对照(Control)(0.92±0.06比1.18±0.05比1.14±0.03、1.42±0.05比1.90±0.12比1.77±0.14、2.02±0.03比2.83±0.08比2.70±0.10、2.78±0.02比3.78±0.03比3.70±0.17、3.22±0.04比4.10±0.12比4.02±0.10，n t24 h＝7.983、n t48 h＝9.024、n t72 h＝23.491、n t96 h＝66.348、n t120 h＝17.114；n t24 h＝8.453、n t48 h＝5.843、n t72 h＝15.813、n t96 h＝13.479、n t120 h＝19.142，n P值均<0.01)，差异均有统计学意义，但后两组间差异无统计学意义。pHB-shWASF3-1组克隆形成率[(25.50±2.00)%]低于pHB-shNC组及Control组[(62.33±2.57)%、(64.17±3.21)%，n F＝204.754]。迁移实验结果显示pHB-shWASF3-1组穿过小室的细胞数低于pHB-shNC组和Control组[(212.33±14.01)个比(357.33±8.02)个比(364.33±9.07)个，n F＝193.200，n P<0.01]，差异均有统计学意义，但后两组间差异无统计学意义。侵袭实验结果显示pHB-shWASF3-1组穿过小室的细胞数低于pHB-shNC组和Control组[(151.33±5.51)个比(212.33±14.01)个比(215.00±6.08)个，n F＝44.269，n P值均<0.01]，差异均有统计学意义，但后两组间的差异无统计学意义。免疫荧光结果可见，WASF3过表达组的红色信号(WASF3染色)明显高于对照组，绿染的肌动蛋白(F-actin)在细胞边缘明显聚集，细胞伪足伸展更明显。n 结论:WASF3在乳腺癌细胞中呈高表达，静默WASF3后可抑制乳腺癌细胞的增殖、克隆形成及迁移侵袭能力；过表达WASF3后可促进乳腺癌细胞F-action蛋白的堆积，促进细胞片状伪足的形成。“,”Objective:To investigate the effects of Wiskott-Aldrich syndrome protein family verprolin-homologous protein 3 (WASF3) on proliferation, migration, invasion and the formation of pseudopodia in breast cancer cells.Methods:Western blotting was used to detect the expression of WASF3 protein in breast cancer cells (BT-549, HCC-1937, BT-474, MDA-MB-231, MCF-7, T-47D) and normal breast cells (MCF-10A). MDA-MB-231 was selected for gene silencing, and a stable transgenic cell line MDA-MB-231 with WASF3 silencing was constructed through lentivirus infection. The recombinant vector SB-16-WASF3 with WASF3 cDNA and pHB-U6-MCS-PGK-PURO-shWASF3 (pHB-shWASF3) with WASF3 shRNAs were constructed respectively. The lentiviruses were packaged and infected into MDA-MB-231 cells. The WASF3 mRNA and protein expression was verified by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting respectively. The clone formation and migration invasion assays were performed to evaluate cell survival and migration ability, respectively. The SB-16-WASF3 was transfected into MDA-MB-231 cells by liposome. The overexpression of WASF3 and F-actin arrangement were observed via WASF3 antibody and Phalloidin staining.One-way analysis of variance was used for these results, LSD-n t test was used for further pairwise comparison, and n t-test was used for pairwise comparison.n Results:Western blotting results showed that the relative expression of WASF3 in the 6 kinds of breast cancer cells was 1.07±0.00, 0.54±0.11, 0.60±0.14, 1.36±0.02, 0.65±0.05 and 0.79±0.18, respectively, which were significantly higher than that in MCF-10A cells (0.38±0.01, n t=134.864, 21.289, 24.883, 55.397, 8.892 and 37.363, respectively, n P<0.05). Among them, the WASF3 expression in MDA-MB-231 and BT-549 cells was relatively high. The cell proliferation assay showed that the proliferation rate in the stable cells with WASF3 silencing was significantly lower than that in pHB-shNC group and control group at 24, 48, 72, 96 and 120 h (0.92±0.06 vs. 1.18±0.05 vs. 1.14±0.03, 1.42±0.05 vs. 1.90±0.12 vs. 1.77±0.14, 2.02±0.03 vs. 2.83±0.08 vs. 2.70±0.10, 2.78±0.02 vs. 3.78±0.03 vs. 3.7±0.17, 3.22±0.04 vs. 4.10±0.12 vs. 4.02±0.10,n t24 h=7.983, n t48 h=9.024, n t72 h=23.491, n t96 h=66.348, n t120 h=17.114; n t24 h=8.453, n t48 h=5.843, n t72 h=15.813, n t96 h=13.479, n t120 h=19.142, n P<0.01). There was no significant difference between pHB-shNC group and control group. The clone formation rate in WASF3 stable silencing cells [(25.5±2.00)%] was significantly lower than that in pHB-shNC group [(62.33±2.57)%] and control group [(64.17±3.21)%,n F=204.754, n P<0.01]. The results of migration and invasion experiments showed that the number of cells passing through the chamber in pHB-shWASF3-1 group was significantly less than that in pHB-shNC group and control group (212.33±14.01 vs. 357.33±8.02 vs. 364.33±9.07,n F=193.204, n P<0.01; 151.33±5.51 vs. 212.33±14.01 vs. 215.00±6.08,n F=44.269, n P<0.01), and there was no significant difference between the latter two groups. Besides, the immunofluorescence results showed that the F-actin protein gathered at the cell edge and pseudopodia was also formed obviously.n Conclusion:WASF3 is relatively over-expressed in breast cancer cells. WASF3 silencing can inhibit the proliferation, clone formation, migration and invasion of breast cancer cells. WASF3 overexpression can promote the accumulation of F-action protein at the edge of breast cancer cells as well as the formation of pseudopodia.