SPHK1靶向NF-κB信号通路调控胃癌细胞的迁移与侵袭

doi:10.12122/j.issn.1673-4254.2024.11.13

摘要/Abstract

摘要：

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。

关键词: 胃癌, 鞘氨醇激酶-1, NF-κB, 迁移, 侵袭

Abstract:

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.

Key words: gastric cancer, sphingosine kinase-1, NF-κB, migration, invasion

凌潜龙, 纪凯, 陈金业, 管佳佳, 王睿朋, 满文江, 朱冰. SPHK1靶向NF-κB信号通路调控胃癌细胞的迁移与侵袭[J]. 南方医科大学学报, 2024, 44(11): 2163-2171.

Qianlong LING, Kai JI, Jinye CHEN, Jiajia GUAN, Ruipeng WANG, Wenjiang MAN, Bing ZHU. Sphingosine kinase-1 regulates migration and invasion of gastric cancer cells via targeting the nuclear factor-κB signaling pathway[J]. Journal of Southern Medical University, 2024, 44(11): 2163-2171.

图/表 8

表1 SPHK1在GC中的表达与临床病理特征的关系

Tab.1 Clinicopathological characteristics of GC patients (n=40)

Characteristic	Clinicopathological characteristics
Characteristic	n	Percentage (%)
Gender
Male	28	70
Female	12	30
Age (year)
≥60	24	60
<60	16	40
Tumor size (cm)
≥5.0	22	55
<5.0	18	45
Clinical stage
I+II	14	35
III+IV	26	65
N stage
N0	10	25
N1+N2+N3	30	75

表2 RNA干扰和过表达序列

Tab.2 Sequences for RNA interference or gene overexpression

Item	Sequences (5'-3')
shNC	GAGTCTATGACATTGCCTCA
shSPHK1#1	CTTCGTGTCAGATGTTGGATAT
shSPHK1#2	GCTTTGCCCTCACCCTTACAT
shSPHK1#3	GCTTTGCCCTCACCCTTACAT
oeNC	CGCATCTAGCCTGTCAGTCC
oeSPHK1#1	CTCGTGTCAGATATTGGTTAT
oeSPHK1#2	ATTGTGTGAGACATCCGTAAG
oeSPHK1#3	TTGAGTCCTGCTTCTTCATTG

图1 SPHK1在GC组织中的表达

Fig.1 Expression of SPHK1 in GC tissues. A: Expression of SPHK1 in pan-cancer based on data retrieved from TIMER2.0 database. B: Expression of SPHK1 and MKI67 in GC tissues based on data retrieved from GEPIA database. C: GEPIA database analysis of the correlation between SPHK1 and MKI67. D, E: Immunohistochemical staining for SPHK1 and MKI67 in GC tissues from HPA database. *P<0.05, **P<0.01, ***P<0.001 vs Tumor group.

图2 SPHK1表达与GC患者预后的关联

Fig.2 Association of SPHK1 expression with overall survival (OS; A), post-progression survival (PPS; B) and recurrence-free survival (RFS; C) of GC patients predicted using Kaplan-Meier Plotter database.

图3 GC组织中SPHK1表达的上调

Fig.3 SPHK1 expression is upregulated in GC tissue. A: Immunohistochemical staining for detecting SPHK1 and MKI67 in clinical tissue samples (Original magnification:×200). B: Immunohistochemical score of SPHK1 and MKI67. ***P<0.001. C: Correlation between SPHK1 and MKI67 expressions. D: Western blotting for detecting the expression of SPHK1 protein. E: qRT-PCR for detecting the level of SPHK1 mRNA. *P<0.05, **P<0.01, ***P<0.001 vs GES-1 group.

图4 SPHK1对GC细胞迁移与侵袭的影响

Fig.4 Effect of SPHK1 knockdown or overexpression on GC cell migration and invasion. A: Western blotting for assessing efficiency of lentivirus-mediated SPHK1 knockdown and overexpression. B: Western blotting for detecting MKI67 protein levels. C, E: Cell scratch assay for assessing migration ability of the cells (×40). D, F: Transwell assays for assessing invasion ability of the cells (×200). G: Size of the subcutaneous tumors in nude mice. F: Weight of the subcutaneous tumors. *P<0.05, **P<0.01, ***P<0.001 vs NC group.

图5 SPHK1靶向NF-κB信号通路

Fig.5 SPHK1 targets the NF-κB signaling pathway. A: Results of GO enrichment analysis. B: Results of KEGG pathway enrichment analysis. C, E: Expression levels of NF-κB signaling pathway proteins after SPHK1 knockdown. D, F: Expression levels of the proteins after SPHK1 overexpression. *P<0.05, **P<0.01, ***P<0.001 vs NC group.

图6 阻断NF-κB信号通路, SPHK1对GC细胞的迁移与侵袭能力的影响

Fig.6 Effect of blocking the NF-κB signaling pathway on migration and invasion ability of GC cells with SPHK1 knockdown or overexpression. A, B: Transwell assay for assessing migration and invasion of GC cells treated with 100 nmol/L PDTC (a NF-κB pathway inhibitor) or DMSO for 24 h (×200). C-F: Western blotting for detecting the levels of P65, p-P65, VEGFA and IL-17 proteins.*P<0.05, **P<0.01, ***P<0.001.

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