WISP-2 in human gastric cancer and its potential metastatic suppressor role in gastric cancer cells mediated by JNK and PLC-γ pathways

Background:

It has recently been shown that WISP proteins (Wnt-inducted secreted proteins), a group of intra- and extra-cellular regulatory proteins, have been implicated in the initiation and progression of a variety of tumour types including colorectal and breast cancer. However, the role of WISP proteins in gastric cancer (GC) cells and their clinical implications have not yet been elucidated.

Methods:

The expression of WISP molecules in a cohort of GC patients was analysed using real-time quantitative PCR and immunohistochemistry. The expression of a panel of recognised epithelial–mesenchymal transition (EMT) markers was quantified using Q-PCR in paired tumour and normal tissues. WISP-2 knockdown (kd) sublines using ribozyme transgenes were created in the GC cell lines AGS and HGC27. Subsequently, several biological functions, including cell growth, adhesion, migration and invasion, were studied. Potential pathways for the interaction of EMT, extracellular matrix and MMP were evaluated.

Results:

Overexpression of WISP-2 was detected in GC and significantly correlated with early tumour node-metastasis staging, differentiation status and positively correlated with overall survival and disease-free survival of the patients. WISP-2 expression was inversely correlated with that of Twist and Slug in paired samples. Kd of WISP-2 expression promoted the proliferation, migration and invasion of GC cells. WISP-2 suppressed GC cell metastasis through reversing EMT and suppressing the expression and activity of MMP9 and MMP2 via JNK and ERK. Cell motility analysis indicated that WISP-2 kd contributed to GC cells'' motility and can be attenuated by PLC-γ and JNK small inhibitors.

Conclusions:

Increased expression of WISP-2 in GC is positively correlated with favourable clinical features and the survival of patients with GC and is a negative regulator of growth, migration and invasion in GC cells. These findings suggest that WISP-2 is a potential tumour suppressor in GC.     

Aquaporin 5 promotes the proliferation and migration of human gastric carcinoma cells

Aquaporin 5 (AQP5) promotes the progression and invasion of several cancers, but its role in the tumorigenesis of human gastric carcinoma (GC) has not been clearly defined. Here, we investigated the potential functions of AQP5 in the proliferation and migration of human GC. RT-PCR and western blotting were used to detect the expression of AQP5 in human GC cell lines. Immunohistochemistry was applied to evaluate the expression of AQP5 in human GC tissues and corresponding normal tissues. Following ectopic overexpression of AQP5 or inhibition of AQP5 by its inhibitor, acetazolamide (AZA), cell proliferation and migration of AGS cells were analyzed by MTT assay, colony formation assay, and wound healing assay. Heterogeneous expression of AQP5 mRNA and protein was observed in human GC cell lines MKN45, MKN28, AGS, and SGC7901. AQP5 was up-regulated in GC tissues in comparison to corresponding normal tissues. AQP5 protein was mainly localized in the cell membrane. Overexpression of AQP5 was correlated with enhanced lymph node metastasis. In vitro, overexpression of AQP5 notably enhanced, while inhibition of AQP5 by AZA significantly attenuated the proliferation and migration of AGS cells. Our data indicate that AQP5 may play an important role in the tumorigenesis and progression of human GC and suggest that AQP5 is a potential therapeutic target against GC.     

miR-26a靶向MMP16参与调控胃癌细胞侵袭作用研究

背景与目的：胃癌发生侵袭转移是导致患者预后不良的重要因素。本研究旨在明确miR-26a在调控胃癌细胞运动侵袭中的作用及其可能机制。方法：通过实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)检测胃癌组织细胞中miR-26a表达情况,体外通过CCK-8法、平板克隆形成实验和Matrigel-Transwell实验评价miR-26a对人胃癌细胞增殖、运动和侵袭能力的影响。荧光素酶报告基因系统评估miR-26a对下游靶基因的调控作用。结果：胃癌组织中miR-26a表达较癌旁组织明显下降。miR-26a体外对胃癌细胞增殖无明显影响,但可显著抑制胃癌细胞的运动和侵袭。相反,抑制miR-26a表达可促进胃癌细胞的侵袭能力。生物信息学分析提示,基质金属蛋白酶16(matrix metalloproteinase 16,MMP16)为miR-26直接调控靶基因,miR-26a可抑制胃癌细胞MMP16 miRNA和蛋白的表达。荧光素酶报告基因检测提示,miR-26a可与MMP16 mRNA 3’UTR结合诱导其转录后抑制。进一步研究提示,MMP16 siRNA对胃癌细胞侵袭具有模拟miR-26a作用,而过表达MMP16可拮抗miR-26a对胃癌细胞侵袭的影响。结论：miR-26a可通过靶向MMP16来抑制胃癌细胞运动侵袭,miR-26a可作为抑制胃癌细胞侵袭的重要干预靶点。     

lncRNA MAFG-AS1通过调控miR-11181-3p/GLG1轴促进胃癌AGS细胞迁移、侵袭和有氧糖酵解

目的：探讨lncRNA MAFG-AS1/miR-11181-3p/GLG1分子轴对胃癌（gastric cancer,GC）细胞迁移、侵袭和有氧糖酵解的影响及其可能的机制。方法：选取 MAFG-AS1 相对高表达的 GC 细胞系 AGS 作为研究对象,采用 qPCR 法检测其 MAFGAS1、miR-11181-3p、GLG1的RNA表达水平,Transwell实验、糖酵解分析等检测细胞迁移、侵袭和有氧糖酵解的变化,利用生物信息学分析及双荧光素酶报告基因验证MAFG-AS1、miR-11181-3p、GLG1之间的相互作用关系。结果：敲减MAFG-AS1显著上调miR-11181-3p及下调GLG1的表达（均P<0.01）,并可显著抑制GC细胞迁移、侵袭和有氧糖酵解（均P<0.01）;荧光素酶报告基因证实MAFG-AS1竞争性吸附miR-11181-3p（P<0.01）;抑制miR-11181-3p或过表达GLG1可部分逆转敲减MAFG-AS1对GC细胞迁移、侵袭和有氧糖酵解的抑制作用（均P<0.05或P<0.01）。结论：MAFG-AS1通过miR-11181-3p/GLG1分子轴增强GC迁移、侵袭和有氧糖酵解,可能是GC诊疗的潜在分子靶点。     

miR-875-5p通过靶向USF2抑制胃癌细胞的增殖、迁移和侵袭

目的:探讨miR-875-5p对胃癌细胞增殖、迁移和侵袭的影响及其机制。方法:采用qPCR法检测胃癌细胞BGC-823、HGC-27、MGC-803、SGC-7901、AGS、MKN-45和胃黏膜上皮细胞GES-1中miR-875-5p的表达水平。利用脂质体转染技术,分别将miR-875-5p模拟物/抑制剂(mimic/inhibitor)及其阴性对照质粒(miR-NC/Anti-miR-NC)转染至AGS细胞/MKN-45细胞,构建过表达/抑制miR-875-5p的细胞模型,空白对照组(Control组)不转染。通过CCK-8、克隆形成、Transwell等实验分别检测miR-875-5p表达变化对细胞增殖、克隆形成、迁移和侵袭的影响。采用双荧光素酶报告基因实验验证miR-875-5p与上游刺激因子2(USF2)的靶向关系,WB实验验证miR-875-5p对USF2的调控作用并检测USF2蛋白的表达。构建MKN-45细胞裸鼠移植瘤模型,验证miR-875-5p过表达对MKN-45细胞成瘤能力的影响。结果:miR-875-5p在6种胃癌细胞中表达水平显著低于胃黏膜上皮细胞GES-1(均P<0.01)。与Control组和miR-NC组相比,miR-875-5p mimic组AGS细胞的增殖、克隆形成率、迁移和侵袭细胞数,以及USF2蛋白的表达均显著降低(P<0.05或P<0.01);miR-875-5p inhibitor组MKN-45细胞的增殖、克隆形成率、迁移和侵袭细胞数,以及USF2蛋白的表达均显著提高(P<0.05或P<0.01)。双荧光素酶报告基因实验证明,miR-875-5p能够直接靶向USF2基因。体内成瘤实验结果表明,过表达miR-875-5p显著抑制MKN-45细胞移植瘤的生长(均P<0.01)。结论:miR-875-5p通过靶向USF2抑制胃癌细胞的增殖、迁移和侵袭。     

蒿甲醚对胃癌细胞增殖、侵袭和迁移的抑制作用研究

目的:研究蒿甲醚对胃癌细胞（AGS和SGC-7901）的增殖、侵袭以及迁移的影响并探讨其分子机制。方法:分别利用MTT、集落形成试验检测胃癌细胞的活力和生长;流式细胞技术检测胃癌细胞的凋亡率;利用细胞侵袭和划痕愈合试验检测胃癌细胞的侵袭和迁移;Western blot检测相关蛋白的表达水平。结果:蒿甲醚（0~800 μmol/L）能够浓度和时间依赖性的抑制 AGS和SGC-7901细胞的增殖（P＜0.05）。集落形成试验以及流式细胞术结果显示蒿甲醚（400 和 600 μmol/L）能够抑制AGS和SGC-7901细胞的生长以及促进细胞凋亡（P＜0.05）。细胞侵袭和划痕愈合试验发现蒿甲醚（400 和 600 μmol/L）能够抑制AGS和SGC-7901细胞的侵袭和迁移（P＜0.05）。Western blot结果显示蒿甲醚(400 和 600 μmol/L)能够增加AGS和SGC-7901细胞的cleaved caspase-3,cleaved caspase-9以及Bax的蛋白水平（P＜0.05）;同时能够抑制N-cadherin 和vimentin的蛋白表达并促进E-cadherin蛋白的表达。结论:蒿甲醚可以显著抑制胃癌细胞的增殖、侵袭以及迁移,其机制可能是与促进细胞凋亡以及抑制上皮间质转化有关。     

锌指蛋白883在胃癌组织中的表达及生物学功能

目的:探讨锌指蛋白883（ZNF883）在胃癌组织中的表达及预后意义,并观察其对胃癌细胞增殖、迁移和侵袭的影响。方法:基于TCGA数据,通过GEPIA网络平台分析ZNF883 mRNA在胃癌组织和正常胃组织中的表达差异及其与患者生存率的相关性。通过蛋白免疫印记（Western blotting,WB）检测ZNF883蛋白在胃癌组织及对应癌旁组织中的表达水平。ZNF883 shRNA转染人胃癌细胞AGS和NCI-N87,WB检测敲低效率,CCK-8和Transwell小室检测细胞增殖、迁移和侵袭,并通过WB检测细胞周期蛋白D1（CCND1）、细胞周期依赖性激酶4（CDK4）和基质金属蛋白酶2/9（MMP2/9）蛋白表达。结果:TCGA数据分析发现ZNF883 mRNA在胃癌组织中的表达显著高于正常胃组织,其蛋白表达在胃癌组织中亦显著上调。生存分析证实ZNF883 mRNA高表达胃癌患者的无病生存率和总生存率均明显降低。敲低ZNF883显著抑制AGS和NCI-N87细胞增殖、迁移和侵袭。另外,敲低ZNF883显著减少胃癌细胞中CCND1、CDK4和MMP2/9蛋白水平。结论:ZNF883是一个新的胃癌驱动基因,胃癌组织中其高表达提示患者预后不良,ZNF883可能通过促进CCND1、CDK4和MMP2/9表达增强胃癌细胞增殖、迁移和侵袭。     

Regulation of migration and invasion by Toll-like receptor-9 signaling network in prostate cancer

Toll-like receptors (TLRs) play an important role in tumorigenesis and progress of prostate cancer. However, the function and mechanism of Toll-like receptor-9 (TLR9) in prostate cancer is not totally understood. Here, we found that high expression of TLR9 was associated with a higher probability of lymph node metastasis and poor prognosis. Further in vitro functional study verified that silence of TLR9 inhibited migration and invasion of PC-3 cells, indicating expression of TLR9 involving in the migration and invasion of cancer cells. The data of microarray exhibited silence of TLR9 induced 205 genes with larger than 2-fold changes in expression levels, including 164 genes down-regulated and 41 genes up-regulated. Functional Gene Ontology (GO) processes annotation demonstrated that the top three scores of molecular and cellular functions were regulation of programmed cell death, regulation of locomotion and response to calcium ion. TLR9 signaling network analysis of the migration and invasion related genes identified several genes, like matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 9 (MMP9), chemokine receptor 4 (CXCR4) and interleukin 8 (IL8), formed the core interaction network based on their known biological relationships. A few genes, such as odontogenic ameloblast-associated protein (ODAM), claudin 2 (CLDN2), gap junction protein beta 1 (GJB1) and Rho-associated coiled-coil containing protein kinase 1 pseudogene 1 (ROCK1P1), so far have not been found to interact with the other genes. This study provided the foundation to discover the new molecular mechanism in signaling networks of invasion and metastasis in prostate cancer.     

Curcumin inhibits tongue carcinoma cells migration and invasion through downregulation of matrix metallopeptidase 10

Squamous cell carcinoma (SCC) of tongue is an aggressive head and neck cancer with high propensity of regional spreading and invasion. Tongue carcinoma cells treated with curcumin, the major curcuminoid of the turmeric, demonstrated reduction in adhesion, migration, and invasion ability. High-throughput microarray analysis indicated that curcumin treatment suppressed matrix metallopeptidase 10 (MMP10) expression. MMP10 is overexpressed in tongue carcinoma tissues in comparison with the normal epithelia. Curcumin treatment on tongue carcinoma cell lines suppressed MMP10 expression at both mRNA and protein levels. Our results suggested that curcumin is a promising inhibitor to tongue cancer cells migration and invasion.     

RANKL/RANK通路介导神经母细胞瘤细胞迁移的机制探讨

目的:探讨RANKL/RANK通路对神经母细胞瘤（neuroblastoma,NB）SH-SY5Y细胞系细胞侵袭和转移的作用机制。方法:通过pcDNA3.1+-RANKL和siRNA-RANKL转染NB SH-SY5Y细胞系过表达或沉默外源基因RANKL;细胞增殖实验(cell counting kit-8,CCK-8)检测外源基因RANKL转染NB SH-SY5Y细胞系对细胞增殖的影响;划痕试验检测外源基因RANKL转染NB SH-SY5Y细胞系对细胞迁移的影响;Western blot实验检测外源性基因RANKL转染NB SH-SY5Y细胞系后细胞内基质金属蛋白酶9（matrix metalloproteinase 9,MMP9）、基质金属蛋白酶2（matrix metalloproteinase 2,MMP2）表达变化。结果:通过pcDNA3.1+-RANKL和siRNA-RANKL对NB SH-SY5Y细胞系转染外源性RANKL基因后三组NB细胞增殖能力无统计学差异;RANKL基因过表达后NB SH-SY5Y细胞迁移能力增强（P<0.01）,RANKL沉默后NB SH-SY5Y细胞迁移能力减弱（P<0.01）;RANKL基因过表达后NB SH-SY5Y细胞内MMP9、MMP2表达增强（P<0.05、P<0.001）,RANKL沉默后NB SH-SY5Y细胞内MMP9、MMP2表达减弱（P<0.05、P<0.01）。结论:RANKL/RANK通路介导NB细胞迁移,并可能通过抑制细胞内MMP2、MMP9表达实现。     

G蛋白偶联受体激酶3在口腔鳞状细胞癌细胞增殖、迁移和侵袭中的作用及其可能的机制

目的:探讨沉默G蛋白偶联受体激酶3(G protein-coupled receptor kinase 3,GRK3)对口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)细胞增殖、迁移和侵袭的影响及其可能的机制.方法:利用Oncomine数据库分析GRK3在正常口腔组织及OSCC组织中...     

Transforming growth factor‐β induces CD44 cleavage that promotes migration of MDA‐MB‐435s cells through the up‐regulation of membrane type 1‐matrix metalloproteinase

CD44, a transmembrane receptor for hyaluronic acid, is implicated in various adhesion‐dependent cellular processes, including cell migration, tumor cell metastasis and invasion. Recent studies demonstrated that CD44 expressed in cancer cells can be proteolytically cleaved at the ectodomain by membrane type 1‐matrix metalloproteinase (MT1‐MMP) to form soluble CD44 and that CD44 cleavage plays a critical role in cancer cell migration. Here, we show that transforming growth factor‐β (TGF‐β), a multifunctional cytokine involved in cell proliferation, differentiation, migration and pathological processes, induces MT1‐MMP expression in MDA‐MB‐435s cells. TGF‐β‐induced MT1‐MMP expression was blocked by the specific extracellular regulated kinase‐1/2 (ERK1/2) inhibitor PD98059 and the specific phosphoinositide 3‐OH kinase (PI3K) inhibitor LY294002. In addition, treatment with SP600125, an inhibitor for c‐Jun NH₂‐terminal kinase (JNK), resulted in a significant inhibition of MT1‐MMP production. These data suggest that ERK1/2, PI3K, and JNK likely play a role in TGF‐β‐induced MT1‐MMP expression. Interestingly, treatment of MDA‐MB‐435s cells with TGF‐β resulted in a colocalization of MT1‐MMP and CD44 in the cell membrane and in an increased level of soluble CD44. Using an electric cell‐substrate impedance sensing cell‐electrode system, we demonstrated that TGF‐β treatment promotes MDA‐MB‐435s cell migration, involving MT1‐MMP‐mediated CD44 cleavage. MT1‐MMP siRNA transfection‐inhibited TGF‐β‐induced cancer cell transendothelial migration. Thus, this study contributes to our understanding of molecular mechanisms that play a critical role in tumor cell invasion and metastasis. © 2009 Wiley‐Liss, Inc.     

TMEM16A overexpression contributes to tumor invasion and poor prognosis of human gastric cancer through TGF-β signaling

TMEM16A is a newly identified calcium activated chloride channel, and has been reported to be overexpressed by various solid malignant cancers to promote proliferation and invasion, yet little is known about its role in gastric cancer(GC). Therefore, we investigated the role of TMEM16A in GC and its clinical significance by a retrospective analysis of 367 GC patients, and in vitro study was performed for validation and underlying molecular mechanism.TMEM16A was significantly upregulated and amplified in GC tissues, and its overexpression was positively correlated with disease stage, negatively with patient survival and identified as an independent prognostic factor for patient outcome. A negative correlation between TMEM16A and E-cadherin was found in 367 GC specimens. TMEM16A silencing significantly decreased calcium activated chloride currents, impaired TGF-β secretion, reduced E-cadherin expression, and inhibited the migration and invasion without affecting proliferation of GC cells (AGS and BGC-823). Supplement of TGF-β reverted the effects of TMEM16A silencing on E-cadherin expression, cell migration and invasion.In conclusion, TMEM16A promotes invasion and metastasis in GC, and might be a novel prognostic biomarker and potential therapeutic target in the treatment of GC.     

Connective tissue growth factor enhances the migration of gastric cancer through downregulation of E-cadherin via the NF-κB pathway

Local invasion and distant metastasis are difficult problems for surgical intervention and treatment in gastric cancer. Connective tissue growth factor (CTGF/CCN2) was considered to have an important role in this process. In this study, we demonstrated that expression of CTGF was significantly upregulated in clinical tissue samples of gastric carcinoma (GC) samples. Forced expression of CTGF in AGS GC cells promoted their migration in culture and significantly increased tumor metastasis in nude mice, whereas RNA interference-mediated knockdown of CTGF in GC cells significantly inhibited cell migration in vitro. We disclose that CTGF downregulated the expression of E-cadherin through activation of the nuclear factor-κappa B (NF-κB) pathway. The effects of CTGF in GC cells were abolished by dominant negative IκappaB. Collectively, these data reported here demonstrate CTGF could modulate the NF-κappaB pathway and perhaps be a promising therapeutic target for gastric cancer invasion and metastasis.     

LINC00511靶向miR-497-5p调控胃癌细胞增殖、迁移和侵袭及机制研究

目的:探讨LINC00511对胃癌细胞增殖、迁移和侵袭的影响及其作用机制。方法:将pcDNA、pcDNA-LINC00511、si-NC、si-LINC00511、miR-NC、miR-497-5p分别转染至MGC-803细胞中,分别记为pcDNA组、pcDNA-LINC00511组、si-NC组、si-LINC00511组、miR-NC组、miR-497-5p组;将si-LINC00511质粒分别与anti-miR-NC、anti-miR-497-5p共转染至MGC-803细胞中,分别记为si-LINC00511+anti-miR-NC组、si-LINC00511+anti-miR-497-5p组。实时荧光定量PCR（RT-qPCR）检测miR-497-5p和LINC00511表达水平;蛋白质印迹（Western Blot）法检测细胞周期素D1（cyclin D1,CyclinD1）、p21、基质金属蛋白酶2（matrix metalloproteinase 2,MMP2）、基质金属蛋白酶9（matrix metalloproteinase 9,MMP9）蛋白表达水平;四甲基偶氮唑盐比色法(MTT)检测细胞活性;Transwell检测细胞迁移和侵袭;双荧光素酶报告基因实验检测LINC00511和miR-497-5p的靶向关系。结果:与正常胃黏膜上皮细胞GES-1相比,胃癌细胞MGC-803、MKN-45、AGS中miR-497-5p表达水平显著降低,LINC00511表达水平显著升高。LINC00511靶向调控miR-497-5p的表达。抑制LINC00511表达和miR-497-5p过表达可降低细胞活性和迁移、侵袭数量,降低CyclinD1、MMP2、MMP9蛋白表达水平,提高p21蛋白表达水平。干扰miR-497-5p表达逆转了抑制LINC00511表达对胃癌MGC-803细胞增殖、迁移和侵袭的抑制作用。结论:抑制LINC00511表达可抑制胃癌细胞增殖、迁移和侵袭,其机制可能与miR-497-5p表达有关,将为胃癌的治疗提供新思路和新靶点。     

miR-6775-3p在乳腺癌细胞中的表达及其对乳腺癌细胞生物学行为的影响

背景与目的：微小RNA（microRNA,miRNA）与肿瘤的发生、发展过程密切相关。探讨miR-6775-3p在乳腺癌细胞系中的表达及其对乳腺癌细胞生物学行为的影响。方法：通过实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）检测4种乳腺癌细胞系MDA-MB-231、MDA-MB-453、MDA-MB-468和BT-549中miR-6775-3p的表达水平,选取miR-6775-3p表达水平最低的乳腺癌细胞系过表达miR-6775-3p后,采用细胞计数试剂盒（cell counting kit-8,CCK-8）法检测细胞的增殖情况,同时采用transwell迁移和侵袭实验分别检测细胞迁移和侵袭能力的变化。通过RTFQ-PCR和蛋白［质］印迹法（Western blot）检测miR-6775-3p过表达的乳腺癌细胞系中细胞周期蛋白依赖性蛋白激酶4（cyclin-dependent protein kinase 4,CDK4）和CDK6,以及侵袭转移标志物基质金属蛋白酶（matrix metalloproteinase,MMP）17和MMP24 mRNA以及蛋白的表达变化。结果：RTFQ-PCR结果显示,乳腺癌细胞系MDA-MB-453中miR-6775-3p的表达最低,在MDA-MB-453细胞中转染miR-6775-3p mimics后,miR-6775-3p的表达水平明显升高（P<0.001）。CCK-8实验结果显示,MDA-MB-453细胞过表达miR-6775-3p后,细胞的增殖能力明显降低（P<0.01）。Transwell迁移和侵袭实验结果显示,MDA-MB-453细胞过表达miR-6775-3p后,细胞的迁移（P<0.001）和侵袭能力（P<0.01）明显降低。RTFQ-PCR和Western blot实验结果显示,CDK4、CDK6及MMP17、MMP24的mRNA表达和蛋白水平均显著降低（P<0.01）。结论：miR-6775-3p可能抑制乳腺癌细胞的增殖、迁移和侵袭能力。     

CXCR4 is a prognostic marker that inhibits the invasion and migration of gastric cancer by regulating VEGF expression

Metastasis is the main cause of poor prognosis of patients with gastric cancer (GC). Thus, current research is focused on identifying biomarkers that can predict the prognosis of patients with GC. C-X-C motif chemokine receptor 4 (CXCR4) and vascular endothelial growth factor (VEGF) have been reported to play important roles in different types of malignancies; however, their role in the prognosis of GC remains unknown. The present study aimed to investigate the potential role of CXCR4 and VEGF in predicting the prognosis of patients with GC. Immunohistochemistry analysis was performed to analyze the expression levels of CXCR4 and VEGF in a GC tissue microarray containing GC tissues and adjacent normal tissues. The association between CXCR4 or VEGF expression levels and the clinicopathological characteristics or survival outcomes were assessed. Furthermore, Transwell and wound healing assays were performed to determine the cell invasive and migratory abilities in vitro. The results demonstrated that CXCR4 promoted AGS cell invasion and migration by regulating VEGF expression. In addition, CXCR4 and VEGF expression levels were significantly upregulated in GC tissues compared with adjacent normal tissues, which was associated with a poorer overall survival (OS). Cox regression analysis demonstrated that both upregulated CXCR4 and VEGF expression were independent negative biomarkers of OS. To the best of our knowledge, the present study was the first to discover that CXCR4 and VEGF exert synergistic roles as efficient prognostic indicators for patients with GC.