过表达FOXO1对骨肉瘤细胞侵袭迁移能力的影响及机制研究

［摘要］ 目的 探讨FOXO1对骨肉瘤细胞侵袭迁移能力的影响及机制。方法 采用实时荧光定量PCR（qRT?PCR）的方法检测人正常成骨细胞hFOB 1.19和人骨肉瘤细胞U2OS、MNNG/HOS中FOXO1的mRNA表达水平。利用pcDNA3.1?FOXO1重组质粒建立FOXO1过表达的细胞模型，空载质粒（pcDNA3.1?vector）作为对照，qRT?PCR和蛋白质印迹法（Western blot）验证转染效率。确定成功过表达FOXO1基因后利用Cell Counting Kit?8（CCK?8）实验检测骨肉瘤细胞的增殖能力（吸光度值），Transwell实验体外检测骨肉瘤细胞的侵袭和迁移能力，并采用qRT?PCR和western blot的方法检测基质金属蛋白酶9（MMP9）的表达变化。结果 人骨肉瘤细胞MNNG/HOS、U2OS中FOXO1的mRNA的表达水平显著低于人成骨细胞hFOB 1.19（P<0.05），且骨肉瘤细胞U2OS的FOXO1的表达水平较低。重组质粒pcDNA3.1?FOXO1转染后，骨肉瘤细胞U2OS的FOXO1表达水平显著升高。与对照组（pcDNA3.1?vector）比较，U2OS?FOXO1过表达组的细胞的增殖能力降低（P<0.05），侵袭迁移和迁移能力降低，MMP9较对照组表达水平明显降低（P<0.05）。结论 骨肉瘤细胞中FOXO1表达水平明显低于正常成骨细胞。过表达FOXO1可能通过下调MMP9抑制骨肉瘤细胞的侵袭和迁移能力。

Effect of overexpression of FOXO1 on invasion and migration of osteosarcoma cells and its mechanism

［Abstract］ Objective To investigate the effect of FOXO1 on invasion and migration of osteosarcoma cells. Methods Real?time quantitative PCR （qRT?PCR） was used to detect the mRNA expression level of FOXO1 in human osteoblast hFOB 1.19 and human osteosarcoma cells U2OS and MNNG/HOS. The FOXO1 overexpressed cell model was established by the recombinant plasmid of pcDNA3.1?FOXO1， and the empty plasmid （pcDNA3.1?vector） was used as a control. The transfection efficiency was verified by the method of qRT?PCR and Western blot.The proliferation ability （absorbance value） of osteosarcoma cells was detected by Cell Counting Kit?8 （CCK?8） test after the successful overexpression of FOXO1 gene. The invasion and migration ability of osteosarcoma cells was detected in Transwell experiment in vitro， and the matrix metalloproteinase 9 was detected by qRT?PCR and western blot methods. Results The expression level of FOXO1 mRNA in MNNG/HOS and U2OS of human osteosarcoma cells was significantly lower than that of human osteoblast hFOB 1.19 （P<0.05）， and the expression level of FOXO1 in osteosarcoma cell U2OS was much lower. After transfection of recombinant plasmid pcDNA3.1?FOXO1， the expression level of FOXO1 in osteosarcoma cell U2OS increased significantly. Compared with the control group （pcDNA3.1?vector）， the proliferation of U2OS?FOXO1 overexpressed cells decreased （P<0.05）， and the ability of invasion and migration and migration decreased， and the expression level of MMP9 was significantly lower than that of the control group （P<0.05）. Conclusion The expression level of FOXO1 in osteosarcoma cells is significantly lower than that in normal osteoblasts. Overexpression of FOXO1 may inhibit the invasion and migration of osteosarcoma cells by downregulating MMP9.