Calcium release-activated calcium channel modulator 1 promotes the migration and invasion of SW480 colon cancer cell line
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摘要:
目的 探讨钙释放激活钙通道调节分子1（calcium release-activated calcium channel modulator 1，ORAI1）对SW480迁移和侵袭的影响及其机制。 方法 以ORAI1干扰慢病毒感染SW480细胞，用RT-qPCR和Western blot检测细胞中ORAI1mRNA和蛋白的表达，Transwell小室、黏附实验、血管形成及拟态分别检测细胞侵袭、迁移能力，同、异种细胞间黏附及血管生成能力，激光共聚焦显微镜检测细胞钙内流（store-operated Ca2+ entry，SOCE），Western blot法检测细胞中ERK1/2、p-ERK1/2、MMP-2、VEGF和E-cadherin蛋白的表达。 结果 SW480转染ORAI1干扰慢病毒72h后，可见明显的荧光表达；较空病毒组和（或）对照组，干扰组ORAI1的表达降低（P < 0.01）；侵袭和迁移能力减弱（P < 0.01）；同种黏附能力增强（P < 0.05）；异种黏附能力减弱（P < 0.05）；血管生成能力减弱（P < 0.01）；SOCE内流峰值降低（P < 0.05）；p-ERK1/2、MMP-2和VEGF的表达降低（P < 0.01）、E-cadherin表达增加（P < 0.01）。 结论 ORAI1可以促进SW480的迁移和侵袭，其机制可能与SOCE增加有关。 Abstract:Objective To explore the effect of calcium release-activated calcium channel modulator 1 (ORAI1) on the migration and invasion of colon cancer cell line SW480 and its mechanism. Methods The SW480 cells were infected with ORAI interference lentivirus. The expression of ORAI1 mRNA and protein was confirmed by quantitative real-time polymerase chain reaction and Western blot. Transwell chamber, adhesion, angiogenesis, and vasculogenic mimicry experiments were conducted to detect the ability of cell invasion, migration, and angiogenesis and the intercellular adhesion of homogeneous and heterogeneous cells among each group. Confocal microscopy was employed to detect the difference of store-operated Ca2+ entry (SOCE) in each group. Western blott was used to detect the expression of ERK1/2, p-ERK1/2, MMP-2, VEGF, and E-cadherin protein. Results After the infection of SW480 with the ORAI1 interference lentivirus for 72 h, significant fluorescence expression was observed. Compared with the empty vector group and control group, the expression of ORAI1 was lower in the interference group (P < 0.01). Invasion and migration ability decreased (P < 0.01); the intercellular adhesion ability of homogeneous cells increased (P < 0.05); the intercellular adhesion ability of heterogeneous cells decreased (P < 0.05); the angiogenesi and vasculogenic mimicry were enhanced (P < 0.01); the internal flow peak of SOCE was low (P < 0.05); the expression of p-ERK1/2, MMP-2, and VEGF proteins decreased (P < 0.01); and the expression of E-cadherin protein increased (P < 0.01). Conclusion ORAI1 may promote the migration and invasion of SW480. This mechanism may be associated with the increase of SOCE. -
图 1 重组慢病毒感染SW480的荧光图像 （×200）
Figure 1. Fluoroscopic image of SW480 infected with recombinant lentivirus(×200)
A:Interference group; B:Empty virus group
图 2 3组细胞ORAI1表达变化
Figure 2. Expression change of ORAI1 in the three groups of cells
1:Control group; 2:Empty virus group; 3:Interference group
A:Expression of ORAI1 protein; B:mRNA expression of the ORAI1 gene (P < 0.01 compared with control group)
图 3 3组细胞侵袭能力比较 （×200）
Figure 3. Comparison of the invasion ability among the three groups of cells(×200)
图 4 3组细胞迁移能力比较 （×200）
Figure 4. Comparison of the migration ability among the three groups of cells(×200)
图 5 转染细胞标记钙离子探针Rhod-2AM的荧光图像（×400）
Figure 5. Fluoroscopic image of infected cells marked by Ca2+ indicator Rhod-2Am(× 400)
A:Fluoroscopic image of lentivirus; B:Fluoroscopic image of indicator; C:Combined
图 6 3组细胞实时Ca2+荧光强度变化
Figure 6. Changes in real-time Ca2+ fluorescence intensity of three groups of cells
CG: Control group; EG: Empty virus group; IG: Interference group
图 7 各组细胞ERK1/2、p-ERK1/2、MMP-2、VEGF和E-cadherin蛋白的表达变化
Figure 7. Changes in protein expression of ERK1/2, P-ERK1/2, MMP-2, VEGF, and E-cadherin among the groups
表 1 同种及异种细胞间黏附率的比较 （x±s）
Table 1. Comparison between the intercellular adhesion rates of the homogeneous and heterogeneous cells(x±s)
表 2 各组间血管形成与血管拟态比较 （x±s）
Table 2. Comparison of angiogenesis and vascular mimicry among the groups(x±s)
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