Liu L, Li X, Shi J, Li L, Wang J, Luo Z Z
Deparment of Respiration, Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430015, China.
Department of Neurobiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhonghua Zhong Liu Za Zhi. 2018 Sep 23;40(9):659-666. doi: 10.3760/cma.j.issn.0253-3766.2018.09.004.
To investigate the effects of formyl peptide receptor 2 (FPR2) silencing on the proliferation, migration and invasion of human glioma U87 cells and its possible mechanisms. The expression of FPR2 was detected in normal glial cells, glioma cells, normal brain tissues and glioma tissues using Western blot and immunohistochemistry staining. A synthesized siRNA duplex was employed to inhibit FPR2 in human glioma cells (U87). The knockdown efficiency was evaluated by real-time reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot. MTT, transwell assays and flow cytometry analyses were used to determine the cell proliferation, migration, invasion and apoptotic rates of U87 cells, respectively. Mice xenograft experiments were used to observe the effect of FPR2 silencing on the tumorigenesis of U87 cells in vito. Western blot and enzyme-linked immunosorbent assays were performed to detect the expression and release of cell cycle and migration-related proteins. The expression of FPR2 was significantly higher in glioma cell lines and glioma tissues than that in normal glial cells and brain tissues. Compared with blank control and negative control, FPR2 mRNA and protein levels in siRNA group were significantly downregulated. The cell proliferation inhibitory rates in FPR2 siRNA group were (23.1±5.1)%, (39.6±5.6)% and (44.4±6.7)% at 24 h, 48 h and 72 h, respectively, which were significantly increased than those in negative control group [(3.2±0.6)%, (5.7±0.8)% and (7.9±0.9)%, respectively; <0.05]. The apoptosis rate in FPR2 siRNA group was (17.4±2.1)%, which was significantly elevated than that in the negative control group with (5.4±0.5)% and blank control group with (3.8±0.3)% (all <0.05). In addition, the numbers of migrated cells were 108.7±9.5 in FPR2 siRNA group, which was significantly lower than that in blank control group 312.9±17.5 and negative control group (304.4±15.7, all <0.05). Likewise, the numbers of invaded cells were 19.3±3.2 in FPR2 siRNA group, which was significantly lower than that in blank control group 106.9±8.5 and negative control group (102.4±7.4, all <0.05). Moreover, the growth of FPR2 siRNA transfected U87 cells in vivo was remarkably decreased comparing with the negative group (<0.05). Furthermore, the expression of cyclin D1 and VEGF in FPR2 silencing U87 cells was suppressed mainly through β-catenin signaling pathway. FPR2 silencing by siRNA can inhibit the growth, migration and invasion ability, but promote the apoptosis of U87 cells. The possible mechanisms might be associated with the inhibitory expression of cyclin D1 and VEGF.
探讨甲酰肽受体2（FPR2）沉默对人胶质瘤U87细胞增殖、迁移和侵袭的影响及其可能机制。采用蛋白质免疫印迹法和免疫组织化学染色法检测正常神经胶质细胞、胶质瘤细胞、正常脑组织和胶质瘤组织中FPR2的表达。采用合成的小干扰RNA双链体抑制人胶质瘤细胞（U87）中的FPR2。通过实时逆转录-聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法评估敲低效率。分别采用MTT法、Transwell实验和流式细胞术分析来测定U87细胞的增殖、迁移、侵袭及凋亡率。采用小鼠异种移植实验观察FPR2沉默对U87细胞体内成瘤的影响。进行蛋白质免疫印迹法和酶联免疫吸附测定以检测细胞周期和迁移相关蛋白的表达和释放。FPR2在胶质瘤细胞系和胶质瘤组织中的表达明显高于正常神经胶质细胞和脑组织。与空白对照组和阴性对照组相比，小干扰RNA组中FPR2的mRNA和蛋白水平均显著下调。FPR2小干扰RNA组在24 h、48 h和72 h时的细胞增殖抑制率分别为（23.1±5.1）%、（39.6±5.6）%和（44.4±6.7）%，显著高于阴性对照组[分别为（3.2±0.6）%、（5.7±0.8）%和（7.9±0.9）%；P<0.05]。FPR2小干扰RNA组的凋亡率为（17.4±2.1）%，显著高于阴性对照组的（5.4±0.5）%和空白对照组的（3.8±0.3）%（均P<0.05）。此外，FPR2小干扰RNA组迁移细胞数为108.7±9.5，显著低于空白对照组的312.9±17.5和阴性对照组的（304.4±15.7，均P<0.05）。同样，FPR2小干扰RNA组侵袭细胞数为19.3±3.2，显著低于空白对照组的106.9±8.5和阴性对照组的（102.4±7.4，均P<0.05）。此外，与阴性组相比，FPR2小干扰RNA转染的U87细胞在体内的生长明显减慢（P<0.05）。此外，FPR2沉默的U87细胞中细胞周期蛋白D1和血管内皮生长因子的表达主要通过β-连环蛋白信号通路受到抑制。小干扰RNA沉默FPR2可抑制U87细胞的生长、迁移和侵袭能力，但促进其凋亡。其可能机制可能与细胞周期蛋白D1和血管内皮生长因子的表达受抑制有关。
