Objective: To investigate the role of sphingosine kinase-1 (SPHK1) in regulating migration and invasion of gastric cancer (GC) cells.
Methods: TIMER2.0, GEPIA and HPA databases were used to investigate SPHK1 expression in GC, and its association with prognosis of the patients was analyzed using Kaplan-Meier Plotter database. In 40 clinical GC and adjacent tissue samples, SPHK1 and MKI67 expressions were detected with immunohistochemistry, Western blotting, and RT-qPCR. Gene enrichment pathway analysis was conducted to explore the biological functions of SPHK1. In HGC-27 and MGC-803 cells, the effects of lentivirus-mediated SPHK1 knockdown or overexpression on cell migration and invasion and expressions of key proteins in the nuclear factor-κB (NF-κB) signaling were evaluated using cell scratch test, Transwell assays and Western blotting. The changes in tumorigenic capacity of the transfected GC cells were evaluated in nude mice.
Results: SPHK1 was highly expressed in GC tissues in negative correlation with overall survival, overall survival after progression, and relapse-free survival of the patients (all P<0.001). In clinical GC samples, SPHK1 and MKI67 expressions showed a positive correlation (P= 0.00049) and were both significantly up-regulated (P<0.001). Gene enrichment pathway analysis suggested the involvement of SPHK1 in cell adhesion, migration, angiogenesis and the NF-κB pathway (P<0.05). In the cell experiment, SPHK1 knockdown significantly decreased while SPHK1 overexpression enhanced migration and invasion abilities of the GC cells. SPHK1 positively regulated the expressions of phosphorylated P65 (P-P65), VEGFA and IL-17, and blocking the NF-κB pathway by PDTC significantly lowered migration and invasion ability of the cells. In nude mice, the GC cells with SPHK1 knockdown resulted in significantly reduced tumor size and mass, while the SPHK1-overexpressing cells showed enhanced tumorigenicity.
Conclusion: SPHK1 regulates migration and invasion of GC cells via the NF-κB signaling pathway and may serve as a potential diagnostic marker for GC progression.
目的: 探讨鞘氨醇激酶-1（SPHK1）在胃癌（GC）组织中的表达及其靶向核因子-κB（NF-κB）调控GC细胞迁移和侵袭能力的分子机制。
方法: 基于TIMER2.0、GEPIA与HPA数据库分析SPHK1在GC组织中的表达。使用Kaplan-Meier Plotter数据库预测SPHK1与GC患者预后的关联。利用IHC检测GC及癌旁组织中SPHK1和MKI67的表达并分析两者相关性。运用Western blotting与qRT-PCR检测GC各细胞系中SPHK1蛋白及mRNA水平。基因富集通路数据库检索SPHK1对GC进展的生物学功能。使用慢病毒敲低HGC-27/过表达MGC-803细胞中SPHK1的表达；采用细胞划痕实验探究SPHK1对GC细胞迁移能力的影响；Transwell实验探究SPHK1对GC细胞迁移和侵袭能力的作用；通过Western blotting检测各蛋白表达情况。体内成瘤实验中，将裸鼠随机分为shNC组、shSPHK1组、oeNC组与oeSPHK1组，利用稳转株验证SPHK1的促癌作用。
结果: 生物信息学表明SPHK1在GC组织中显著高表达（P<0.001）；同时高表达的SPHK1预示着较差的总生存期（P<0.001）和进展后总生存期（P<0.001）以及更差的无复发生存期（P<0.001）。IHC结果表明GC组织中SPHK1与MKI67表达明显上调（P<0.001）且呈正相关（P<0.001）。基因富集通路数据库提示，SPHK1参与GC中的细胞黏附、迁移及血管生成等，且NF-κB参与GC进展（P<0.05）。细胞实验数据显示，抑制SPHK1减弱GC细胞的迁移和侵袭能力，而过表达SPHK1会产生相反的结果（P<0.01）；SPHK1正向调节磷酸化P65 （p-P65）、血管内皮生长因子（VEGFA）和白细胞介素（IL-17）蛋白表达（P<0.05）；利用PDTC阻断NF-κB信号通路可削弱SPHK1促进的GC细胞迁移与侵袭能力以及各蛋白表达水平（P<0.01）；动物实验表明，与NC组相比，shSPHK1组肿瘤大小和质量明显减小，而oeSPHK1组显著增加（P<0.001）。
结论: SPHK1可靶向NF-κB信号通路表达进而调控GC细胞的迁移与侵袭，提示SPHK1可能是GC进展的潜在诊断分子标志物。
Keywords: NF-κB; gastric cancer; invasion; migration; sphingosine kinase-1.