共刺激分子B7-H3在骨肉瘤细胞侵袭转移中的作用及机制

Effect of Costimulatory Molecule B7-H3 on Invasion and Metastasis of Osteosarcoma Cells and Related Mechanism

目的检测共刺激分子B7-H3在骨肉瘤细胞侵袭转移中的作用及机制。方法运用RT-PCR(reverse transcription-polymerase chain reaction)和Western blot方法检测B7-H3分子在三种骨肉瘤细胞U-2OS、MG-63及Saos-2中的表达。通过Lipofectamine&#174;2000体外转染B7-H3 si RNA或B7-H3 c DNA质粒至骨肉瘤细胞,RT-PCR和Western blot检测细胞中B7-H3 m RNA和蛋白的表达。Transwell侵袭小室实验检测过表达/沉默B7-H3分子对骨肉瘤细胞侵袭能力及对MMP-2表达水平的影响。结果共刺激分子B7-H3在骨肉瘤细胞中均组成性表达。转染B7-H3 si RNA基因后,MG-63细胞B7-H3基因表达水平显著低于空载体转染组和未转染组[(0.131±0.002)vs.(0.504±0.007),(0.131±0.002)vs.(0.613±0.013),均P<0.05]。与对照组细胞相比,转染B7-H3 si RNA后MG-63细胞的侵袭力明显下降(P<0.05),基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)分子的表达水平显著下降(P<0.05)。转染B7-H3 c DNA的Saos-2细胞B7-H3基因表达水平显著高于空载体转染组和未转染组,[(0.701±0.008)vs.(0.512±0.007)],[(0.701±0.008)vs.(0.403±0.013)],均P<0.05],Saos-2细胞的侵袭力明显增加(P<0.05),MMP-2分子的表达水平显著增加,与对照组相比,差异具有统计学意义(P<0.05)。结论 B7-H3基因可能通过调控MMP-2分子进而参与调节骨肉瘤的侵袭转移。

Objective To detect the effect of costimulatory molecule B7-H3 on the invasion and metastasis of osteosarcoma cells and related mechanism. Methods The expression of B7-H3 in osteosarcoma cells(U-2OS, MG-63 and Saos-2) were detected by reverse transcription-polymerase chain reaction(RT-PCR) and Western blot. B7-H3 si RNA or B7-H3 c DNA was transfected in vitro into human osteosarcoma cells using Lipofectamine&#174; 2000. The expressions of B7-H3 m RNA and protein in osteosarcoma cells were analyzed by RT-PCR and Western blot. The invasive capacity of osteosarcoma cells and MMP-2 expression were measured using Transwell invasion assay in vitro. Results All tested cultured osteosarcoma cells constitutively expressed B7-H3 m RNA under normal conditions. After transfection, the level of B7-H3 expression was decreased in B7-H3 si RNA transfected MG-63 cells, compared with control si RNA transfected group[(0.131±0.002) vs.(0.504±0.007), P<0.05] and untransfected group[(0.131±0.002) vs.(0.613±0.013), P<0.05]. Compared with untransfected cells, the invasive ability and the level of matrix metalloproteinase-2(MMP-2) expression of MG-63 cells were decreased significantly by si RNA interference(P<0.05). After transfection of B7-H3 c DNA, the level of B7-H3 expression was increased in transfected Saos-2 cells, compared with vector transfected group[(0.701±0.008) vs.(0.512±0.007), P<0.05] and untransfected group[(0.701±0.008) vs.(0.403±0.013), P<0.05]. Meanwhile, the invasive ability and the level of matrix metalloproteinase-2(MMP-2) expression of Saos-2 cells were increased significantly after B7-H3 c DNA transfection(P<0.05). Conclusion B7-H3 plays a novel role in the regulation of the invasive and metastatic capacity of osteosarcoma cells by regulating MMP-2 expression.