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Hindawi Journal of Oncology Volume 2019, Article ID 4035460, 13 pages https://doi.org/10.1155/2019/4035460 Research Article ANXA2 Silencing Inhibits Proliferation, Invasion, and Migration in Gastric Cancer Cells 1 1 1 1 1 1 Rui Xie, Jia Liu, Xuefeng Yu, Chunfeng Li, Yufeng Wang, Wei Yang, 1 1 1 2 2 2 Jiahe Hu, Ping Liu, Hong Sui, Peiqiang Liang, Xinyan Huang, Lijuan Wang, 1 1 2 1 Yuxian Bai, Yingwei Xue , Jiuxin Zhu , and Tianyi Fang Harbin Medical University Cancer Hospital, Haping Road, Harbin , China Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Medicine Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin , China Correspondence should be addressed to Jiuxin Zhu; email@example.com and Tianyi Fang; firstname.lastname@example.org Received 29 November 2018; Revised 15 March 2019; Accepted 28 March 2019; Published 2 May 2019 Academic Editor: om Th as E. Adrian Copyright © 2019 Rui Xie et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Annexin A2 (ANXA2) has been well known to associate with the progress of malignant tumor. However, the biological behavior of ANXA2 in gastric cancer (GC) remains unclear. We made a hypothesis in transcriptome level from TCGA datasets. en, Th we used immunohistochemical staining to quantify the expression level of ANXA2 protein in GC tissues compared with adjacent tissues. Quantitative real-time PCR and western blot were used for analyzing ANXA2 expression in human GC (SGC-7901, MKN- 45, BGC-823, and AGS) cell lines. We investigated the effect of a lentivirus-mediated knock-down of ANXA2 on the proliferation, invasion and migration of gastric cancer AGS cells. Cell proliferation was examined by MTT and colony formation tests. Cell apoptosis and cycle were measured by flow cytometry. Migration and invasion were detected by transwell assay. We found that high expression of ANXA2 can increase the mobility of cancer cells from TCGA datasets. ANXA2 was upregulated in GC tissues compared with adjacent tissues. AGS cell line displayed significantly higher expression of ANXA2 among the four GC cell lines. In addition, ANXA2 silencing led to a weakened ability of proliferation, invasion, and migration in GC cells; targeting of ANXA2 may be a potential therapeutic strategy for GC patients. 1. Introduction of ANXA2 was observed in many different cancer types, including hepatoma , pancreatic cancer , breast cancer Gastric cancer (GC) is the h highest incidence malignant , glioma , colorectal cancer , and GC . ANXA2 tumor in the world and the third dominant cause of cancer mainly participates in cell membranes formation and takes death, with a 5-year survival rate of only 20% to 25% effect on regulate cytoskeleton. The cytoskeleton changes are worldwide [1, 2]. Despite the improvement of therapeutic common in malignant transformation, adhesion, movement, methods with surgical resection, chemotherapy, radiother- and metastasis which may promote tumor cell to move . apy, immunotherapeutic strategies, and targeted therapies, u Th s, targeting cancerous cells motility may be important to invasion and metastasis lead to the poor prognosis of GC the treatments. However, it is difficult to investigate the exact patients and have become a significant clinical challenge function of ANXA2 in GC directly. With the development [3–7]. er Th efore, finding new molecular markers which are of transcriptomics, analysis of transcriptome sequencing data related to metastasis and poor outcome may contribute to from real pathological specimens may provide a compre- ao ff rding new insights into diagnostic decision and novel hensive background for us to understand the relationship therapies for GC patients. between ANAX2 and GC cells. ANXA2 is a 36 kDa calcium-dependent phospholipid- To clarify this phenomenon, we analyzed the ANAX2 binding cytoskeletal protein; it is also named as Annexin expression in GC tissues from The Cancer Genome Atlas II, Annexin a2, p36, and lipocortin II . Upregulation (TCGA) database by R2 analysis platform. Gene Correlation fift 2 Journal of Oncology analysis, Gene Ontology (GO) analysis, and Kyoto Ency- The primer sequences were as follows: 5 -GTGGTGGAG- clopedia of Genes and Genomes (KEGG) analysis were ATGACTGAAGCC-3 (sense) and 5 -CCACGGGGACTG- performed at the level of transcriptomics. The expression TTATTCG-3 (antisense) were for ANXA2 (110 bp). Glyc- level of ANXA2 protein in GC tissues compared with adjacent eraldehyde 3-phosphate dehydrogenase (GAPDH) was used tissues was evaluated. en, Th we focused on the motility as an internal control; 5 -TGACTTCAACAGCGACAC- changes of GC cells when inhibiting ANXA2, including the CCA-3 (sense) and 5 -CACCCTGTTGCTGTAGCCAAA- function of proliferation, invasion, and migration. 3 (antisense) were for GAPDH (121 bp). The thermal cycling conditions consisted of 1 cycle at 95 Cfor 30s,followed by 45 ∘ ∘ cycles at 95 Cfor 5sand60 C for 30 s. Data were analyzed by 2. Materials and Methods the comparative threshold (2-Ct) method. .. Bioinformatics Analysis. The RNA sequencing (RNA-seq)datawas downloadedfrom TCGAdatabase . Western Blot. Proteins in cell lysates were separated using (https://portal.gdc.cancer.gov/); it contained ANXA2 RNA sodium dodecyl sulfate-polyacrylamide gel electrophoresis expression data for human GC profiles, including 415 tissue and electrophoretically transferred to polyvinylidene samples. Gene Correlation analysis was performed with difluoride membranes. Proteins were probed overnight at the R2: Genomics Analysis and Visualization Platform 4 CwithANXA2rabbit monoclonal antibody andGAPDH (http://r2.amc.nl); the genes and pathways associated with mouse monoclonal antibody (internal standard, Boster Ltd., ANXA2 were tested by GO and KEGG pathway analysis. Wuhan, China), followed by incubation with a horseradish Correlation statistics were calculated using the R2 platform peroxidase-conjugated secondary antibody (Kangcheng and P<0.05 was statistically significant. Bio-tech, China). Antibody binding was detected using peroxidase-conjugated secondary antibodies (Boster Ltd., .. Tissue Microarray. A GC tissue microarray (HStm- Wuhan, China) for another 2 h at room temperature. The Ade180Sur-06) was obtained from Shanghai Outdo Biotech. immunoreactive bands were visualized by western blot GC was staged according to the WHO classification criteria: detection system ECL (Pierce Biotechnology, Rockford, IL, 90 tumor tissues with stages I to IV and paired noncancerous USA) and the intensity of the detected bands was analyzed tissues. eTh present study was approved by the Institutional using an Image J program. Review Boards of participating institutions. .. Construction of Lentivirus and Infection. To knock .. Immunohistochemistry. Immunohistochemical staining down ANXA2 in AGS cells, a small hairpin RNA (shRNA) for ANXA2 was performed by using the EnVision Detec- sequence, 5 -GGATGCTTTGAACATTGAA-3,was tion Systems (Dako, Glostrup, Denmark) according to the designed to target the ANXA2 gene and subcloned manufacturer’s instructions, with ANXA2 rabbit monoclonal into the GV115 lentiviral vector (GeneChem Co., Ltd., antibody (Cell Signaling Technology, Beverly, MA, USA) at Shanghai, China). The scrambled siRNA sequence (5 - 1:2000 dilution as the primary antibody. Staining intensity TTCTCCGAACGTGTCACGT-3 ) was used as the negative of ANXA2 was graded as follows: negative staining (-),<5% control. Subsequently, the lentiviral transfer vector plasmid, of total cells; weak staining (+), 5%-15%; moderate staining packaging plasmid, and envelope plasmid were transfected (++), 16%-50%; and diffuse positive staining (+++), >50%. into human embryonic kidney (HEK) 293T cells (American Type Culture Collection, Manassas, VA, USA) using .. Cell Culture. GC cell lines including SGC-7901, MKN- Lipofectamine 2000 reagent (Invitrogen Life Technologies, 45, BGC-823, and AGS cells with different grade of differen- Carlsbad, CA, USA) for 48 h. Then, cell supernatants tiation were cultured in RPMI1640 medium supplemented containing lentivirus was harvested and concentrated. eTh with 10% fetal bovine serum, 100 U/ml penicillin, and 0.1 virustiter wascalibratedinthe HEK293Tcells.Thelentivirus mg/ml streptomycin, in humidified air containing 5% CO2 with a n fi al concentration of 4 ×10 TU/ml was stored at at 37 C. eTh culture medium was replaced every other day. -80 C. For lentivirus infection, according to the multiplicity of infection (MOI), cells grown to 30% confluence were transfected with lentivirus containing green uo fl rescent .. RNA Isolation and Reverse Transcription. Total RNA was protein (GFP) reporter. eTh cell medium was changed to extracted from cells using TRIzol Reagent (Invitrogen, Carls- complete medium aer ft 12 h of transfection. After cultured bad, CA, USA) according to manufacturer’s protocol. RNA for another 3 d, the infection rates were determined using concentration and quality were determined by the NanoDrop fluorescence microscope. 2000c Spectrophotometer (Thermo Scientific, MA, USA). Subsequently, cDNA was synthesized using M-MLV reverse transcriptase, dNTPs, RNase inhibitor (Promega, Madison, .. Cell Proliferation Assay. Cell proliferation was tested by WI, USA), and an oligo dT purchased from Sangon Biotech evaluating cell viability using the MTT assay. Briefly, cells (Shanghai, China). seeded on 96-well plates were stained with 20𝜇lof5mg/ml MTT dye (Beijing Dingguo Changsheng Biotechnology Co., . . Quantitative PCR (q-PCR). eTh n, q-PCR was carried Ltd., Beijing, China) per well for 4 h at 37 C. After removal of out using SYBR Master Mixture (TaKaRa, Ohtsu, Japan) and the culture medium, 150𝜇l of dimethyl sulphoxide (DMSO, TaKaRa eTh rmal Cycler Dice Real-Time PCR System TP800. Sinopharm Chemical Reagent Co., Ltd., Beijing, China) was Journal of Oncology 3 added to solubilize the formazan. eTh absorbance values were Table 1: Positive correlated genes with ANXA2. determined at a wavelength of 495 nm in a BioTek ELx800 Gene R-value P-value microplate reader. Three replicate wells were measured per ANXA2P2 0.910 <0.005 assay, each experiment was performed at least 3 times. S100A6 0.683 <0.005 S100A16 0.682 <0.005 .. Colony Formation Assay. Cellswereplatedon6-well S100A11 0.637 <0.005 plates at an initial density of 800 cells per plate. eTh cells GPRC5A 0.627 <0.005 were cultured for up to 14 d or allowed to grow until most of colonies reached>50 cells per colony. Aer ft washing with S100A10 0.615 <0.005 phosphate buffered saline (PBS), the colonies were fixed SFN 0.602 <0.005 with 10% paraformaldehyde for 30-60 min, then stained EZR 0.598 <0.005 with Giemsa and washed. Colony number was determined PLEK2 0.593 <0.005 under an Olympus uo fl rescence microscopy equipped with EPHA2 0.583 <0.005 MicroPublisher 3.3 RTV CCD camera. .. Cell Apoptosis Assay. Flow cytometry was performed were takenunderthemicroscope. eTh stainedcells were to detect cell apoptosis rates using Annexin V Apoptosis dissolved in 10% acetic acid and OD 570 was measured. The Detection Kit APC (eBioscience, San Diego, CA, USA) percentage of migration was evaluated by determining the according to the manufacture’s protocol. In brief, aer ft wash- OD570/MTT-OD490 ratio of the cells. ing the collected cells with PBS, the cells were incubated with 5 6 1×Annexin V binding buffer. en Th 100 𝜇lcells (1 ×10 -1×10 .. Wound Scratch Test. Cells in 96-well culture plates cells) were stained with Annexin V-APC for 10 min at room were grown in complete medium until cells reached 90-100% temperature in the dark, we assessed the Annexin V-positive confluence. A wound was scratched across the diameter of cells by a BD FACSCalibur flow cytometer. each plate. The cells were washed gently using PBS to remove cell debris. Cell migration was observed by microscopy at 0, .. Cell Cycle Analysis. Cells in culture were collected, 8, and 24 h. resuspendedinice-coldPBSandfixedwith70% of ice- cold ethanol at 4 C for at least 1 h. Then, the xfi ed wells .. Statistical Analysis. Statistical comparisons between were washed with PBS and stained with propidium iodide groups were determined by Student’s t-test and chi-square (PI, Sigma-Aldrich, St. Louis, MO, USA) and RNase A test using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). Statistical (Fermentas,St. Leon-Rot,Germany)inthedark at room significance was assumed for P<0.05. temperature before analysiing the cell percentage in each cell cycle phase using a FACSCalibur instrument. 3. Results .. Matrigel Invasion Assay and Transwell Migration Assay. .. Expression of ANXAinTCGADatabase. By analyzing Cell invasion assay was tested using Biocoat Matrigel Inva- the expression level of ANXA2 in TCGA database, we found sion Chamber (Becton-Dickinson, Bedford, MA), a 24-well that ANXA2 was highly homogeneously expressed in GC transwell unit with 8𝜇m pore size polyethylene terephthalate tissues (Figure 1). The samples were divided into T stage membrane, according to the manufacturer’s protocol. Briefly, (T1b, 14 samples; T2, 65 samples; T3, 181 samples, T4, 31 1×10 cells in the top chamber incubated in 100𝜇loffetal samples); N stage (N0, 123 samples; N1, 112 samples; N2, 80 bovine serum- (FBS-) free medium were allowed to attach samples; N3, 29 samples); and M stage (M0, 367 samples; M1, to 24-well transwell plates. eTh lower invasion chamber 27 samples). en Th we analyzed the expression of ANXA2 in contained 600𝜇l of RPMI-1640 medium with 30% FBS. Aer ft different stages. We found that the expression level of ANXA2 24 h of incubation, the cells that invaded the lower surface in patients with distant metastasis (M1) was higher than that of the membrane were xfi ed and stained with Giemsa for 30 in nondistant metastasis patients (M0). But there was no min. Under a phase-contrast microscope, we got the images statistical significance in T stage and N stage (Figure 2). of the cells in vfi e predetermined elds fi at a magnification of 100 and 400. The stained cells were dissolved in 10% acetic acid [16, 17]; the invasion was calculated by measuring the .. Function of ANXA and Its Related Genes Analyzed by optical density (OD) values at a wavelength of 570 nm (OD KEGG and GO Analysis. Using the R2 platform analysis, 570). 415 samples were analyzed statistically; 3323 positive genes Cells (1×10 )incubated in100 𝜇lofFBS-freemedium and 5700 negative genes were significantly correlated with were added to the 12-well format top chambers with 8.0 ANXA2 expression. The top 10 positively and negatively 𝜇m pore size porous transparent polyethylene terephthalate correlated genes were shown in Tables 1 and 2. To determine membrane. eTh lower chamber of the 12-well format tran- the function of ANXA2 and its related genes, we used GO swell contained 600𝜇l of RPMI-1640 medium with 30% FBS. analysis and KEGG analysis (Tables 3 and 4). Biological After incubation for 24 h, the cells that invaded the lower processes included structure morphogenesis and movement surface of the membrane were stained with Giemsa. Pictures of cells. Among the 9023 genes associated with ANXA2, 4 Journal of Oncology Tumor Stomach Adenocarcinoma - TCGA 11 11 9 9 Samples ordered by ANXA2 Figure 1: By analyzing the expression of ANXA2, we found ANXA2 was w idely expressed in gastric cancer tissues. eTh blue points showed the expression level ANXA2. Different colors of X-Line indicated gastric cancer patients with different stages. Tumor Stomach Adenocarcinoma - TCGA Tumor Stomach Adenocarcinoma - TCGA Tumor Stomach Adenocarcinoma - TCGA 15.5 15.5 15.5 15.0 15.0 15.0 14.5 14.5 14.5 14.0 14.0 14.0 13.5 13.5 13.5 13.0 13.0 13.0 12.5 12.5 12.5 12.0 12.0 12.0 11.5 11.5 11.5 11.0 10.5 11.0 11.0 n0 (123) n1 (112) n2 (80) n3 (29) t1b (14) t2 (65) t3 (181) t4 (31) m0 (367) m1 (27) Figure 2: Differential expression of ANXA2 could differentiate M staging of gastric cancer patients ( P=0.04), not T staging or N staging. Table 2: Negative correlated genes with ANXA2. of ANXA2 in GC tissues and adjacent tissues based on immunohistochemical analysis. ANXA2 mainly expressed Gene R-value P-value in GC tissues with obvious brownish yellow particles (Fig- CBFA2T2 -0.561 <0.005 ure 4(d)). Negative ANXA2 expression was detected in 29 CHD6 -0.559 <0.005 (32.2%) of the 90 GC tissues samples, 6 samples (6.7%) were ZFHX3 -0.540 <0.005 hadropositive and 25 samples (27.8%) were positive ANXA2 expression (Table 5(a)). The remaining samples were weakly BSN -0.529 <0.005 positive. In contrast to GC tissues, 91.1% of the adjacent tis- NTN3 -0.513 <0.005 sues showed negative ANXA2 expression (Figure 4(c)). The ZNF445 -0.508 <0.005 data showed that the distribution of ANXA2 expression in IQSEC1 -0.505 <0.005 GC and adjacent tissues was statistically significant ( P<0.001, CBX6 -0.486 <0.005 Table 5(b)). SEZ6 -0.481 <0.005 IRF2BPL -0.480 <0.005 .. ExpressionofANXAinDiﬀerentGCCellLines. Expres- sion levelofANXA2mRNAinhumanGCcelllines with different grade of differentiation was examined by quantita- KEGG analysis found that 3006 genes were involved in 47 tive real-time PCR. eTh results showed that ANXA2 mRNA signaling pathways; most of them were related to pathways was positively expressed in four human GC cells (SGC-7901, in cancer and regulation of actin cytoskeleton (P<0.005). BGC-823, MKN-45, and AGS cells), the highest expression Among the genes coexpressed with ANXA2, we found of ANXA2 mRNA was tested in AGS cells (Figure 5(a)). that the signaling pathways ‘Focal adhesion’ and ‘Regulation Consistent with mRNA expression level, AGS cell had the of actin cytoskeleton’ were activated, which was the same highest expression level of ANXA2 protein detected by as we anticipated (Figure 3). This actin polymerization may western blot (Figure 5(b)). change cytoskeleton. On the other hand, the filamentous filopodia may help the movement of tumor cells. ..ExpressionofANXAmRNAandProteininAGSCells. To .. Expression of ANXA in GC Tissues and Adjacent Tissues. suppress ANXA2 expression, AGS cells were infected with a Our experiments summarized the different expression level lentivirus which expressed ANXA2-spcific siRNA and GFP. 2log of ANXA2 2log of ANXA2 2log of ANXA2 2log of ANXA2 2log of ANXA2 Journal of Oncology 5 Table3:GOanalysisofANXA2in thedatabase. Biological process No. of genes P-value Go path no. membrane-bounded organelle 5877 <0.005 43227 anatomical structure morphogenesis 1332 <0.005 9653 cell morphogenesis 553 <0.005 902 protein binding 5369 <0.005 5515 movement of cell or subcellular component 1006 <0.005 6928 cytoskeleton organization 673 <0.005 7010 Table 4: KEGG analysis of ANXA2 in the database. Biological process No. of genes P-value Related genes APPL1, FZD10, LPAR6, GNA13, CXCL12, ABL1, Pathways in cancer 224 <0.005 PIK3R5, AKT3, MTOR, STAT5A, ARAF, BCL2, SMAD4, TGFBR1, MSH3, PIAS2... EGF, EGFR, PIK3R5, ITGA11, LAMC3, PAK4, DOCK1, Focal adhesion 122 <0.005 ROCK1, GRF2, MAPK8, JUN, BLC2, ELK1, BRAF, RAPGEF1, CRK, SOS1... AKT2, AKT3, ANGPT1, ARNT, BCL2, CAMK2A, HIF 1 signaling pathway 67 <0.005 CAMK2B, EGLN1, ENO3, EP300, EPO, PIK3CD, PIK3CG,TEK,TF, VHL... ELK1, ROCK1, TIAM1, ITPR1, DROSHA, HOXD10, Proteoglycans in cancer 116 <0.005 PIK3R5, AKT3, PDPK1, MTOR, PDCD4, COL21A1, ARAF, HGF, FRS2, SOS1, WNT16... F2, GNA13, ARAF, PIK3R5, SOS1, EGFR, EGF, ITGA11, Regulation of actin cytoskeleton 118 <0.005 CHRM1, FGD1, VAV3, MSN, ACTN4, MYLK, MYH9, CFL1, ENAH, WASF1... AKT2, FGFR1, FLT3, GCK, KIT, MTOR, NRAS, Central carbon metabolism in cancer 42 <0.005 NTRK1, NTRK3, PDGFRA, PFKM, PGAM2, PIK3CD, RET... ACTA2, ADRA1A, AGTR1, BRAF, CACNA1C, Vascular smooth muscle contraction 76 <0.005 EDNRA, GNA12, GNAS, ITPR1, KCNMA1, MRVI1, PLA2G12A, PLCB4, RAMP3, ROCK1... ∗For the “Biological process” column, we showed the function of ANXA2 in gastric cancer tissue. The “No. of genes” column showed the number of genes that enriched in corresponding pathways, and the details were in the “Related genes” column. Correlation statistics were calculated by the R2 platform. Table 5: eTh expression of ANXA2 in GC tissues. (a) ANXA2 expression Tissue type Number of cases Negative Weakly positive Positive Hadro-positive (-) (+) (++) (+++) 82 4 3 1 Adjacent 90 (91.1%) (4.4%) (3.3%) (1.1%) ↓ 29 ↑ 20 ↑ 25 ↑ 6 Cancer 90 (32.2%) (22.2%) (27.8%) (6.7%) ∗↓ Decreased ANXA2 expression in gastric cancer tissue compared to adjacent tissue. ↑ Enhanced ANXA2 expression in gastric cancer tissue compared to adjacent tissue. (b) Adjacent Cancer Total Number P-value Negative Total positive Negative 29 28 1 <0.001 Positive 61 54 7 6 Journal of Oncology REGULATION OF ACTIN CYTOSKELETON Chemotactic factor Chemotactic factor INS Bradykinin FN1 LPA CXCL12 F2 Acetylcholine GF LPAR CXCR4 F2R RTK ITG GPCR Sos FAK G G12,13 Cas JNK, p38 MAPK signaling Gene expression CrkII pathway Raf Ras FGD1/3 c-Src +p Dock180 MEK PI3K Rac1GEF +p ０）０3 ERK Actin polymerization Vav/Tiam1 Drf3 IRSp53 Mena F-Actin Arp2/3 Cdc42 Adherens junction IQGAP NWASP +p Filopodia RhoGEF GRLF1 Asef APC Rho Rac PIR121 HSPC300 TMSB4 Nap125 WAVE1 Abi2 PXN GIT1 PIX +p Actin PFN +p +p polymerization NHE1 PAK Focal adhesion ERM IRSp53 WAVE2 assembly ROCK +p VCL PI4P5K ACTN ０）０ 2 +p +p +p +p F-Actin Focal adhesion mDia MLCP MLCK PI4P5K LIMK +p Arp2/3 -p +p Stabilization CFN of actin PFN -p MLC SSH Lamellipodia ０）０ Actin polymerization ? stress ﬁber Actomyosin assembly contraction GSN VCL F-Actin MyosinII Focal complex Focal adhesion Stress ﬁbers Assembly 04810 7/10/18 (c) Kanehisa Laboratories (a) FOCAL ADHESION RhoGAP mDia1 +p +p RhoGEF ROCK MLC RhoA -p Regulation of PIP5K MLCP +p +p +p Cell motility actin cytoskeleton Zyxin MLCK Stress ﬁber / FA Actinin VASP Actin +p formation ０）０ polymerization Filamin ITGA Vinculin ECM Actin ITGB Filopodia Parvin Talin Lamellipodia PDK1 formation Paxillin ECM-receptor Src +p FA turnover interaction +p -Catenin Calpain ILK Akt/PKB GSK-3 +p +p +p PKC +p Wnt signaling Cdc42 pathway PTEN Caveolin Cell proliferation +p -p -p PI3K-Akt FAK PI3K Vav Rac PAK signaling pathway CycD ０）０ DNA Phosphatidyl inositol Fyn signaling system Cell cycle DOCK1 +p +p +p p130Cas GRF2 Rap1 c-Jun Crk JNK C3G Cytokine- cytokine receptor MAPK B-Raf interaction signaling pathway Cell survival +p +p +p +p +p +p +p +p RTK GF Shc Grb2 Sos Ha-Ras Raf-1 MEK1 ERK1/2 Elk1 cIAPs DNA +p +p +p Bad Bcl-2 04510 3/16/18 (c) Kanehisa Laboratories (b) Figure 3: (a) By KEGG pathway enrichment analysis, ANXA2 upregulated filopodia pathway, resulting in cell metastasis. Microfilament is one component of cytoskeleton; it is mainly composed of actin. ANXA2 upregulated regulation of actin cytoskeleton pathway and led to the rise of cell deformability. (b) By KEGG pathway enrichment analysis, ANXA2 upregulated focal adhesion pathway, resulting in cell motility, proliferation and survival. Journal of Oncology 7 Adjacent Cancer Negative Negative Staining Staining (a) (b) Adjacent Cancer ANXA2 ANXA2 (c) (d) Figure 4: Immunohistochemical staining of ANXA2 was upregulated in gastric cancer tissue than adjacent tissue. Negative staining of (a) adjacent tissue and (b) gastric cancer tissue. ANXA2 expression of (c) adjacent tissue and (d) gastric cancer tissue (magnification, 100 ×,scale bar, 50𝜇m). After 72 h infection, more than 80% of cells expressed GFP, assay provided evidence that ANXA2 silencing inhibited indicating successful infection (Figure 5(c)). ANXA2 mRNA invasion and migration ability in ANXA2 knock-down group (Figure 5(d)) and ANXA2 protein expression (Figure 5(e)) than in control and negative control groups (Figures 7(a) and in AGS cells infected with lentivirus expressing ANXA2 7(b)). The results showed that the migration rate of ANXA2 siRNA (knock-down group) were significantly lower than knock-down group cells was significantly decreased at 24 h in cells infected with lentivirus expressing scrambled siRNA than in negative control and control cells (Figures 7(c) and (negative control group) and AGS cells (control group). These 7(d)). results demonstrated that siRNA directed towards ANXA2 was efficient in specifically knocking down the ANXA2 gene 4. Discussion in AGS cells. GC is a heterogeneous disease with a high mortality rate. Its . . e FunctionofANXAonProliferation inAGSCells. As poor prognosis is mainly contributed by extensive invasion illustrated in Figures 6(a) and 6(b), compared with control and metastasis . The mechanism of tumor metastasis is and negative control groups, numbers of cells colony forma- complex. It includes cancerous cells away from their primary tion capacity significantly reduced in ANXA2 knock-down tumor, the intravasation into the bloodstream or lymph group. eTh MTT assay showed obviously less proliferation in nodes, the transit through the tumor microenvironment, and ANXA2 knock-down group at 4 and 5 d aer ft infection than the aggregation to secondary tissues. Cancer cell migration other groups (Figure 6(c)). eTh se n fi dings totally revealed plays an important role in the metastatic process. However, that ANXA2 was essential for AGS cell proliferation. ANXA2 the specicfi reasons have not been clariefi d yet. eTh refore, in- silencing caused G1 phase arrest of AGS cells and a decline depth research on genes related to invasion and metastasis of in the number of cells in the S and G2/m phases compared GC is crucial. to negative control and control cells (Figure 6(d)). The ANXA2 is overexpressed in most tumor tissues and rates of apoptosis in ANXA2-silencing cells were appreciably acts as a pivotal part in tumor development. Prior research increased (Figure 6(e)). generally conrm fi s that the expression level of ANXA2 has a close relation with progression of tumors [19–21]. Zhang . e Function of ANXA on Invasion and Migration in et al.  prove that expression level of ANXA2 mRNA AGS Cells. Matrigel invasion chamber, transwell, and scratch is higher in GC tissues than nontumor tissues. Besides, 8 Journal of Oncology ∗∗ SGC-7901 GES-1 BGC-823 MKN-45 AGS ANXA2 GAPDH SGC-7901 GES-1 BGC-823 MKN-45 AGS (a) (b) Control Negative control Knock down 200× 200× 200× Bright field 200× 200× 200× Fluorescent field (c) 1.2 ∗∗ 1.0 0.8 Negative Knock Control control down 0.6 0.4 ANXA2 0.2 GAPDH 0.0 Control Negative control Knock down (d) (e) Figure 5: ANXA2 siRNA suppressed the ANXA2 mRNA and protein expression levels in gastric cancer cell lines. (a) Quantitative real- time PCR analyzed ANXA2 mRNA levels in different gastric cancer cell lines including SGC-7901, GES-1, BGC-823, MKN-45, and AGS. ∗P<0.05,∗∗P<0.01 compared to GES-1. (b) eTh expression level of ANXA2 protein in SGC-7901, GES-1, BGC-823, MKN-45, and AGS cells was determined using western blot. GAPDH was utilized as the internal control. (c) Bright and GFP uo fl rescent field of AGS cell 72 h aer ft infection with negative control (middle panel) or lentivirus containing ANXA2-RNAi (knock-down, right panel), magnification, 200 ×,scale bar, 100𝜇m. (d) Quantitative real-time PCR assessment of ANXA2 mRNA levels in AGS siRNA-infected cells compared to control and negative control cells,∗∗P<0.01. (e) Western blot analysis of ANXA2 protein expression in ANXA2 knock-down cells compared to control and negative control cells. Relative mRNA level Relative mRNA level (ANXA2/GAPDH) (ANXA2/GAPDH) Journal of Oncology 9 Control Negative control Knock down Bright field Fluorescent field Giemsa stained (a) 1.6 1.4 1.2 1.0 ∗∗ 0.8 0.6 ∗∗ 0.4 60 0.2 0.0 day 1 day 2 day 3 day 4 day 5 Control Negative control Control Negative control Knock down Knock down (b) (c) Control Negative control Knock down 700 700 350 600 600 300 500 500 250 400 200 300 150 & 200 200 100 100 100 50 0 0 0 0 020 40 60 80 100 120 G1 S G2/M 0 20406080 100120 020 40 60 80 100 120 Control Negative control Knock down (d) Figure 6: Continued. Number Clone OD490 Percentage (%) 10 Journal of Oncology Control Negative control Knock down 200 200 160 160 160 12 120 120 80 80 M1 80 M1 M1 40 40 0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 (e) Figure 6: ANXA2 silencing inhibited AGS cell growth. (a) Representati ve size of cell colonies, bright field (upper panel), uo fl rescent field (middle panel), and Giemsa stained (lower panel), P<0.05. (b) The number of cell colonies in control, negative control, and ANXA2 knock- down cells,∗P<0.05. (c) Using MTT assay, the relative AGS cell proliferation pattern at different time points was investigated, ∗P<0.05, n=5. (d) eTh ratio of cells at different cell cycle phases in control, negative control, and ANXA2 knock-down groups. (e) The cell apoptotic rate in control, negative control, and ANXA2 knock-down groups using flow cytometry, ∗P<0.05, n=3. as the expression level of ANXA2 increases, the degree of Our results observed that the proliferation and colony pathological differentiation of GC cells decreases . Con- formation ability of lentivirus-mediated Annexin A2 knock- sistent with previous study, our research demonstrated that down cells were suppressed appreciably; it prompted us ANXA2 was significantly enhanced in GC tissues compared to consider that ANXA2 might act as a positive role in with adjacent tissues. proliferation of GC cells in vitro. ANXA2 knock-down cells Previous reportshaveshowntheeeff ct ofANXA2in showed a significant more proportion of cells in the G1 phase many cancerous cells. For example, ANXA2 may promote and a decline number in the S and G2/m phases compared theprogressionandinvasionofhuman lungcancer cell to negative control or control cells. Similarly, in previous . With the upregulation of ANXA2, the potential ability study, non-small cell lung cancer cell lines with ANXA2 of metastatic and invasive is increased in hepatoma cell, silencing showed inhibition both in tumor growth and cell shRNA-mediated ANXA2 silencing significantly inhibits cell proliferation and also induced cell cycle arrest at the G2 phase invasion, migration, and tumorigenic potential . Similar . Apoptosis rates apparently increased in ANXA2 knock- studies have studied ovarian cancer cell , glioma cell , down cells. Abnormal acceleration of cell cycle and inhibition and breast cancer cell . However, there is no relevant of apoptosis are important causes of the tumor cell growth. study on biological behavior of ANXA2 in GC cells. These results suggested that ANXA2 was a promising target We first analyzed the expression of ANXA2 in GC tissue for inhibiting GC cell proliferation possible by blocking the from TCGA database;itmadeanewviewfor theresearch cell cycle and promoting cell apoptosis. of GC patients. eTh oretically, the large size of the RNA- Invasion and metastasis as the important biological Seq dataset may improve the reliability of the results. u Th s, behaviors of malignant tumor are the leading causes for data analysis depending on RNA-Seq can provide a more poor prognosis of GC patients . Numerous factors realistic context for us. It was interesting that we first verified contributed to regulate invasion and metastasis and other our conjecture from TCGA database. eTh high expression biological behaviors in tumor cells. Our experiment showed of ANXA2 activated tumor cells to generate cytoskeleton, that knocking down ANXA2 expression inhibited invasion acted a positive role in proliferation of GC cells. On the other and migration ability of AGS cells to achieve the purpose of hand, the high expression of ANXA2 activated the pathway delaying the progression of GC. for tumor cells to grow filopodia, it provided energy for In our previous published study, we used gene expression cancerous cells to move themselves. From this perspective, microarray and detected 100 differentially expressed genes we explained why the high expression of ANXA2 indicated including 58 upregulated and 42 downregulated human poor prognosis of GC. genes. According to all pathway genetic information in In our study, AGS cell line displayed the highest expres- KEGG and BIOCARTA, we did the enrichment analysis sion level of ANXA2 among the four kinds of human GC of the difference of gene; the important ANXA2 related cell lines and it was selected as a cell model for subsequent genes including FGA, FGB, SERPINB2, CD55, PLAUR, MET, experiments. To elucidate roles of ANXA2 in GC cell growth, RAP1A, and ETS1 were screened. Western blot showed that AGS cells were infected with a lentivirus expression ANXA2- aer ft silencing of ANXA2, RAP1A, and MAP2K1 protein spcific siRNA and GFP, it laid the foundation for the further expression decreased in AGS cells. We speculated that RAP1A experiments. and MAP2K1 might be potential downstream regulating Apoptosis (%) Control Negative control Knock down Journal of Oncology 11 Control Negative control Knock down 200× 200× 200× 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Control Negative Knock down control (a) Knock down Control Negative control 0.6 200× 200× 200× 0.4 0.2 0.0 Control Negative Knock control down (b) Control Negative control Knock down 4.6 5.23 5.11 4.15 4.09 4.39 4.72 4.63 4.72 0 h 0 h 0 h 2.32 2.26 2.2 2.95 2.77 2.5 2.23 2.41 2.98 8 h 8 h 8 h 1.03 0.52 1.54 1.6 2.35 0.73 0 24 h 24 h 24 h (c) 1.5 1.0 0.5 0.0 Control Negative Knock down control 8 h 24 h (d) Figure 7: ANXA2 silencing inhibited invasion and migration rate of AGS cells. (a) Giemsa staining and compared with negative control group, theinvasionrateinknock-downgroupwasdecreased,∗P<0.05, n=3, scale bar, 100𝜇m. (b) Giemsa staining and compared with negative control group, the migration rate in knock-down group was decreased,∗P<0.05, n=3, scale bar, 100𝜇m. (c) Picture of cell scratch, scale bar, 100𝜇m. (d) In 8 h, the migration rate has no significant difference in control, negative control, and knock-down groups. Compared with control group and negative control group, at 24 h, the migration rate of knock-down group was lower,∗P<0.05, n=3. Migration rate Migration rate Invasion rate 12 Journal of Oncology genes of ANXA2 taking part in MAPK signaling pathway in accuracy or integrity of any part of the work are appropriately AGS cells . investigated and resolved. Identifying target points of genes related to proliferation, invasion, and migration of GC is essential to consummate Acknowledgments traditional treatments and improve prognosis of patient. Our experimental results provided further insights into This work was supported by the Yu Weihan Academician’s the research progression on biological behavior of GC. It Outstanding Youth Training Fund in Harbin Medical Univer- could lead researchers to taking serious consideration of the sity, Nn10 program (Nn10 PY 2017-03), Youth Elite Training potential benefit of ANXA2 silence as an effective antitumor Fund (JY2015-02), Outstanding Youth Fund (JCQN 2018- therapeutic tool. 04) in Harbin Medical University Cancer Hospital, and Natural Science Foundation of Heilongjiang Province of China (QC2013C077). 5. Conclusions In conclusion, our results presented a compelling evidence References for the role of ANXA2 in biological behavior of GC. Ana-  J. Ferlay, I. Soerjomataram, R. 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