目的:我们旨在研究表皮生长因子样结构域7(EGFL7)对乳腺癌细胞增殖和血管生成的影响及其与p38丝裂原活化蛋白激酶(p38MAPK)信号通路的关系。
方法:构建用于EGFL7稳定过表达的载体和用于EGFL7敲除的载体。本研究选择乳腺癌细胞株MDA-MB-231,将细胞分为4组:对照组,空载体组(用空载体转染),EGFL7过表达组(用EGFL7过表达载体转染),和EGFL7敲除组(用EGFL7敲除载体转染)。转染72小时后,RT-PCR和Westernblot检测细胞中EGFL7的mRNA和蛋白水平,分别。12h时的细胞增殖率,24h,用MTT法检测各组培养48h和72h。建立了肿瘤-内皮细胞共培养体系的体外肿瘤血管生成模型,并在12h,24h,使用体外血管生成测定法比较各组之间的48小时和72小时培养。将EGFL7过表达组的细胞进一步分为三组,分别用p38MAPK抑制剂SB203580以0μmol/L的剂量处理,5μmol/L,10μmol/L,分别。之后,细胞与内皮细胞共培养48h。Westernblot检测血管内皮生长因子(VEGF)蛋白水平,p38MAPK,和p-p38MAPK.
结果:与对照组相比,EGFL7过表达组的EGFL7mRNA水平较高,而EGFL7敲除组的EGFL7mRNA水平较低(均P<0.05)。12h时与对照组比较,24h,48h,和72小时的培养,EGFL基因敲除组细胞增殖率较低,EGFL基因过表达组细胞增殖率较高,(均P<0.05)。此外,在这些时间点与对照组相比,血管芽的数量和VEGF的蛋白质水平,p38MAPK,p-p38MAPK在EGFL7基因敲除组较低,在EGFL7过表达组较高,(均P<0.05)。用SB203580处理过表达EGFL7的细胞后,p-p38MAPK水平下降,VEGF蛋白表达水平与SB203580浓度呈负相关(F=44.24,P<0.01)。
结论:EGFL7可以促进乳腺癌细胞的增殖和血管生成,其机制可能与p38MAPK信号通路的激活和促进VEGF的表达有关。
OBJECTIVE: We aimed to investigate the effects of epidermal growth factor-like domain 7 (EGFL7) on breast cancer cell proliferation and angiogenesis and its association with the p38 mitogen-activated protein kinase (p38MAPK) signaling pathway.
METHODS: The vectors for stable overexpression of EGFL7 and the vectors for EGFL7 knockout were constructed. The breast cancer cell line MDA-MB-231 was selected for this study and the cells were divided into four groups: the control group, the empty vector group (transfected with an empty vector), the EGFL7 overexpression group (transfected with the EGFL7 overexpression vector), and the EGFL7 knockout group (transfected with the EGFL7 knockout vector). After 72 h of transfection, the mRNA and protein levels of EGFL7 in the cells were detected by RT-PCR and Western blot, respectively. The cell proliferation rates at 12 h, 24 h, 48 h and 72 h of culture in each group were detected using the MTT method. An in vitro tumor angiogenesis model of tumor-endothelial cells co-culture system was established and the angiogenesis ability at 12 h, 24 h, 48 h and 72 h of culture were compared among the groups using an in vitro angiogenesis assay. The cells in the EGFL7 overexpression group were further divided into three groups and were treated with p38MAPK inhibitor SB203580 at a dose of 0 μmol/L, 5 μmol/L, and 10 μmol/L, respectively. Afterward, the cells were co-cultured with endothelial cells for 48 h. Western blot was performed to detect the protein levels of vascular endothelial growth factor (VEGF), p38MAPK, and p-p38MAPK.
RESULTS: Compared with the control group, the EGFL7 mRNA level was higher in the EGFL7 overexpression group and lower in the EGFL7 knockout group (both P<0.05). Compared with the control group at 12 h, 24 h, 48 h, and 72 h of culture, the cell proliferation rates were lower in the EGFL knockout group and higher in the EGFL overexpression group, respectively (all P<0.05). Moreover, compared with the control group at these time points, the number of vascular sprouts and the protein levels of VEGF, p38MAPK, and p-p38MAPK were lower in the EGFL7 knockout group and higher in the EGFL7 overexpression group, respectively (all P<0.05). After the cells overexpressing EGFL7 were treated with SB203580, the level of p-p38MAPK was deceased, and the protein expression level of VEGF was inversely related with the SB203580 concentration (F=44.24, P<0.01).
CONCLUSIONS: EGFL7 can promote the proliferation of breast cancer cells and angiogenesis, and the mechanism may be associated with the activation of p38MAPK signaling pathway and promotion of VEGF expression.