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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 midkine (MK) gene on the proliferation, migration, adhesion and invasion of human breast cancer MDA-MB-231 cells. Methods: The mRNA and protein expression of MK in human breast cancer cell lines MCF-7, Bcap-37 and MDA-MB-231 were detected by real-time fluorescence quantitative PCR and Western blotting, and the cell lines with higher expression of MK were screened out. MK shRNA interference plasmid pSilencer-3.1-H1-MK and empty vector control plasmid pSilencer-3.1-H1-NC were transfected into this cell line with lipofectamine 2000, and the untreated control group was set. Real-time fluorescent quantitative PCR and Western blotting were used to detect the expression of MK gene and protein. CCK-8 was used to detect the proliferation of cells and the adhesion ability to ECM was detected after 30 min. Transwell assay was used to detect the invasion and migration of cells. Results: Real-time PCR and Western blotting showed that the MDA-MB-231 cell line was suitable for knockdown validation. The relative expression level of MK gene in MK-MB-231 cells treated with pSilencer-3.1-H1-MK was 0.38 ± 0.02, lower than that in control group (0.76 ± 0.04) and empty vector group (0.84 ± 0.04, F = 144.85, P <0.001; relative expression of MK protein was 0.39 ± 0.07, lower than the control group 0.95 ± 0.04 and empty vector transfection group 0.99 ± 0.02, F = 26.49, P = 0.001. The results of CCK-8 showed that the cell proliferation ability of MK gene knockdown group was significantly lower than that of control group and empty vector transfected group at 24, 48 and 72 hours after cell culture, the difference was statistically significant (P <0.05). The results of cell adhesion assay showed that the number of adherent cells in the MK gene interference group was (21.87 ± 5.17)% after treated with extracellular matrix protein for 30min, which was lower than that in the control group (38.74 ± 4.98)% and the empty vector transfection group (42.37 ± 5.74) )%, F = 27.60, P <0.001. In Transwell migration assay, the number of cells that translocated into Transwell cells by MK gene was 26.6 ± 6.67, lower than that of control group (47.0 ± 4.32) and empty vector transfection group (52.0 ± 6.98), F = 44.98, P <0.001. In the invasion assay, the number of cells passing through the Transwell chamber was 13.2 ± 3.46, lower than that of the control group (19.4 ± 4.43) and the empty vector transfected group (19.9 ± 3.90), F = 8.94, P = 0.001. Conclusion: The expression of MK can significantly inhibit the proliferation, adhesion, migration and invasion of human breast cancer MDA-MB-231 cells in vitro.