摘要 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-t检验,两两比较采用t检验。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,t=134.864、21.289、24.883、55.397、8.892、37.363,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,t_(24 h)=7.983、t_(48 h)=9.024、t_(72 h)=23.491、t_(96 h)=66.348、t120 h=17.114;t_(24 h)=8.453、t_(48 h)=5.843、t_(72 h)=15.813、t_(96 h)=13.479、t120 h=19.142,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� 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-t test was used for further pairwise comparison,and t-test was used for pairwise comparison.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,t=134.864,21.289,24.883,55.397,8.892 and 37.363,respectively,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.
出处 《中华实验外科杂志》 CAS 北大核心 2021年第4期602-605,共4页 Chinese Journal of Experimental Surgery
基金 国家自然科学基金(81572616、81772845) 山东省自然科学基金(ZR2017MH016)。
分类号 R73 [医药卫生—肿瘤]
