摘要
目的:观察中期因子(midkine,MK)基因对人乳腺癌MDA-MB-231细胞增殖、迁移、黏附和侵袭能力的影响。方法:实时荧光定量PCR和蛋白质印迹法检测人乳腺癌细胞株MCF-7、Bcap-37和MDA-MB-231中MK mRNA及蛋白表达,筛选出MK表达丰度较高的细胞株。采用脂质体2000将MK shRNA干扰质粒pSilencer-3.1-H1-MK和空载体对照质粒pSilencer-3.1-H1-NC转染到该细胞株,并设未处理对照组。实时荧光定量PCR和蛋白质印迹法检测干扰后MK基因和蛋白表达;CCK-8检测细胞增殖能力,与胞外基质蛋白作用30min后检测其黏附能力,Transwell检测细胞侵袭能力和迁移能力。结果:实时荧光定量PCR和蛋白质印迹检测结果显示,MDA-MB-231细胞株适合做敲减验证。pSilencer-3.1-H1-MK干扰MDA-MB-231细胞MK表达后,MK基因相对表达量为0.38±0.02,低于对照组0.76±0.04和空载体转染组0.84±0.04,F=144.85,P<0.001;MK蛋白相对表达量为0.39±0.07,低于对照组0.95±0.04和空载体转染组0.99±0.02,F=26.49,P=0.001。CCK-8结果显示,细胞培养24、48和72h,MK基因干扰组细胞增殖能力明显低于低于对照组和空载体转染组,差异有统计学意义,P<0.05。细胞黏附实验结果显示,与胞外基质蛋白作用30min后,MK基因干扰组黏附细胞数为(21.87±5.17)%,低于对照组(38.74±4.98)%和空载体转染组(42.37±5.74)%,F=27.60,P<0.001。Transwell迁移实验中,MK基因干扰组穿越Transwell小室的细胞个数为26.6±6.67,低于对照组(47.0±4.32)和空载体转染组(52.0±6.98),F=44.98,P<0.001。侵袭实验中,MK基因干扰组穿越Transwell小室的细胞个数为13.2±3.46,低于对照组(19.4±4.43)和空载体转染组(19.9±3.90),F=8.94,P=0.001。结论:干扰MK的表达可显著抑制人乳腺癌MDA-MB-231细胞体外增殖、黏附、迁移和侵袭能力。
OBJECTIVE： To investigate the effect of MK on the proliferation, adhesion, migration and invasion of breast cancer cell line MDA-MB-231. METHODS：Real-time PCR and western blot analysis were used to test the mRNA and protein expression levels of MK of human breast cancer cell lines MCF-7, Bcap-37 and MDA-MB-231 ,and MK high- expressed cell were selected. A shRNA plasmid pSilencer-3.1-H1-MK which specifically targets the mRNA of MK and the empty vector pSilencer-3.1-H1-NC were transfected into the selected cell through Lipofectamine 2000,and untreated cells as control group. After transfection,the mRNA and protein expression levels of MK were tested through real-time PCR and western blot analysis. The cell proliferation was evaluated by CCK-8 assay. After incubation with extracellular matrix proteins for 30 rain, the adhesive ability of the cell line was investigated. Transwell systems were used to measure the inva- sion and migration abilities of the seiected cells. RESULTS： Real-time PCR analysis and western blot assay showed that MDA-MB-231 was suitable for interference test. Knockdown of MK resulted in a decrease of MK mRNA expression in MDA-MB-231 cells （0.38±0.02）,as compared with the control group（0.76 ±0.04）, and the empty vector transfection group （0.84±0.04）,F=144.85,P〈0. 001;MK protein expression level （0. 394±0.07） was lower than that in control group（0.95±0.04） and the empty vector transfection group （0.99±0.02） ,F=26.49,P=0. 001. Knockdown of MK ledto decreased cell proliferation at 24 h,48 h and 72 h, as compared with the control group and the empty vector transfection group （P〈0.05）. In addition, knockdown of MK resulted in a decrease of the number of MDA-MB-231 adhesion cells （21.87±5.17） % ,compared with the control group（38.74±4.98）% and the empty vector transfection group （42.37± 5.74） % ,F= 27.60, P〈0. 001. The migration and invasion assay showed that after MK knockdown,the number of MDA- MB-231 cells that passed the transwell membran
出处
《中华肿瘤防治杂志》
CAS
北大核心
2014年第16期1213-1218,共6页
Chinese Journal of Cancer Prevention and Treatment
基金
国家自然科学青年基金(81101493)
