SEMA6D对人骨肉瘤MG63细胞生物学行为的影响△

Effect of SEMA6D on biological behavior of human osteosarcoma MG63 cells

摘要:

[目的] 探讨信号素 6D （semaphorin 6D, SEMA6D） 对骨肉瘤细胞增殖、侵袭的影响及其机制。[方法] 将 MG63 细胞分为空白对照组 （Ctrl 组）、阴性对照组 （si-NC 组） 和转染靶向 SEMA6D 的 si-RNA 组 （si-SEMA6D 组），给予相应的体外转染。采用 CCK-8、细胞划痕、Transwell 侵袭实验检测 MG63 细胞增殖、迁移及侵袭能力的变化。Western blot 检测下游相关信号通路蛋白表达情况。[结果] 培养 24 h，三组 CCK-8 值差异无统计学意义 （P＞0.05），在 48 h 和 72 h，Ctrl、si-NC 组 CCK-8 值显著高于 si-SEMA6D 组 [(0.7±0.1) vs (0.7±0.1) vs (0.4±0.1), P＜0.001; (1.7±0.1) vs (1.6±0.1) vs (1.0±0.1), P＜0.001]。Transwell 侵袭实验显示，Ctrl、si-NC 组细胞侵袭数显著高于 si-SEMA6D 组 [(435.0±28.2) vs (400.7±41.4) vs (291.3±31.1), P=0.022]。划痕 24 h、48 h，Ctrl、si-NC 组划痕愈合率显著高于 si-SEMA6D 组 [(48.8±3.3)% vs (40.6±3.4)% vs (16.6±2.4)%, P＜0.001; (74.7±1.1)% vs (67.6±3.0)% vs (49.5±2.3)%, P＜0.001]。Western blot 检测表明，相较于 si-SEMA6D 组，Ctrl 组、si-NC 组 p-PI3K、p-AKT、pp38、MMP2 的蛋白表达水平均显著增加 （P＜0.05），而三组中 P13K、AKT、p38、Bcl-2 及 Bax 的表达水平差异无统计学意义 （P＞0.05）。[结论] 沉默 SEMA6D 后骨肉瘤 MG63 细胞可通过抑制 PI3K/AKT 及 p38-MAPK 信号通路，抑制细胞增殖、迁移及侵袭能力。

Abstract:

[Objective] To investigate the effects of semaphorin 6D (SEMA6D) on the proliferation and invasion of osteosarcoma cells and its mechanism. [Methods] MG63 cells were divided into blank control group (the Ctrl group), negative control group (the si-NC group) and the group transfected with si-RNA targeting SEMA6D (the si-SEMA6D group), and corresponding transfection was performed in vitro. The changes of proliferation, migration and invasion ability of MG63 cells were detected by CCK-8, cell scratch and Transwell invasion assay. In addition, western blot analysis was performed to detect the protein expression of downstream related signaling pathways. [Results] After 24 hours culture, there was no significant difference in CCK-8 among the three groups (P＞0.05). At 48 hours and 72 hours, the Ctrl and si-NC groups were significantly higher than the si-SEMA6D groups in CCK-8 assay [(0.7±0.1) vs (0.7±0.1) vs (0.4±0.1), P＜0.001; (1.7±0.1) vs (1.6±0.1) vs (1.0±0.1), P＜0.001]. The Ctrl and si-NC groups were significantly higher than the si-SEMA6D group in Transwell invasion assay [(435.0±28.2) vs (400.7±41.4) vs (291.3±31.1), P=0.022]. At 24 hours and 48 hours, the Ctrl and si-NC groups had significantly higher scratch healing rate than the si-SEMA6D group [(48.8±3.3)% vs (40.6±3.4)% vs (16.6±2.4)%, P＜0.001; (74.7±1.1)% vs (67.6± 3.0)% vs (49.5±2.3)%, P＜0.001]. As consequence of western blot analysis, the Ctrl group and si-NC group were significantly increased compared with the si-SEMA6D group in protein expression levels of p-PI3K, p-AKT, p-P38 and MMP2 (P＜0.05), although there were no significant differences in the expression levels of P13K, AKT, p38, Bcl-2 and Bax among the three groups (P＞0.05). [Conclusion] SEMA6D silencing of osteosarcoma MG63 cells can inhibit cell proliferation, migration and invasion by inhibiting PI3K/AKT and p38-MAPK signaling pathways.