Objective: 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. Methods: 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. Results: 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; P<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 P<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 P<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 P<0.05). Moreover, the growth of FPR2 siRNA transfected U87 cells in vivo was remarkably decreased comparing with the negative group (P<0.05). Furthermore, the expression of cyclin D1 and VEGF in FPR2 silencing U87 cells was suppressed mainly through β-catenin signaling pathway. Conclusions: 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.
目的： 探讨沉默甲酰肽受体2(FPR2)基因对胶质瘤U87细胞生长、迁移和侵袭能力的影响及其可能机制。 方法： 采用Western blot法和免疫组化法检测正常胶质细胞、胶质瘤细胞、正常脑组织和胶质瘤组织中FPR2蛋白的表达水平。以小干扰RNA(siRNA)沉默人胶质瘤U87细胞中FPR2基因的表达，采用实时荧光定量PCR和Western blot法检测FPR2 mRNA和蛋白的表达量；采用四甲基偶氮唑蓝(MTT)法检测转染FPR2 siRNA 24、48和72 h后对U87细胞增殖的作用；采用流式细胞术检测FPR2基因沉默对U87细胞凋亡水平的影响。以Transwell小室和裸鼠成瘤实验检测FPR2基因沉默对U87细胞迁移侵袭和成瘤能力的影响。采用Western blot法和酶联免疫吸附实验检测细胞周期和迁移相关蛋白的表达和释放水平。 结果： 与正常胶质细胞和正常脑组织比较，FPR2蛋白在胶质瘤细胞和胶质瘤组织中的表达明显上调。与空白对照组和阴性对照组比较，siRNA干扰组U87细胞中FPR2 mRNA和蛋白的相对表达量明显降低。siRNA干扰组U87细胞在转染24、48和72 h时的生长抑制率分别为(23.1±5.1)%、(39.6±5.6)%和(44.4±6.7)%，与阴性对照组[分别为(3.2±0.6)%、(5.7±0.8)%和(7.9±0.9)%]比较，差异有统计学意义(P<0.05)。siRNA干扰组U87细胞在转染48 h后的凋亡率为(17.4±2.1)%，明显高于阴性对照组[(5.4±0.5)%]和空白对照组[(3.8±0.3)%]；siRNA干扰组迁移细胞数为(108.7±9.5)个，明显低于空白对照组[(312.9±17.5)个]和阴性对照组[(304.4±15.7)个]；siRNA干扰组侵袭细胞数为(19.3±3.2)个，明显低于空白对照组[(106.9±8.5)个]和阴性对照组[(102.4±7.4)个]，差异均有统计学意义(P<0.05)。与阴性对照组比较，siRNA干扰组的裸鼠成瘤能力明显降低。siRNA干扰可明显下调细胞周期蛋白cyclin D1的表达和血管内皮生长因子(VEGF)的表达和释放，其主要通过β－catenin信号通路来实现。 结论： siRNA沉默FPR2基因可抑制胶质瘤U87细胞的增殖、迁移和侵袭，并通过下调cyclin D1的表达和抑制VEGF的水平来实现。.
Keywords: FPR2; Glioma; Invasion; Proliferation; U87 cells.