The Expression Of TNFAIP8 In Human Gastric Cancer And Its Effect On Regulation Of Cell Proliferation, Invasion And Migration

Background:Gastric cancer(GC), although decreasing in incidence within the past few years, continues to be one of the serious threats to human health. Worldwide, GC is an aggressive tumor leading to the fourth human malignancy and the second cause of death. Although surgical resection and adjuvant chemotherapy had made great progress and some GC can be cured at the early stage, unfortunately, most patients were in an advanced stage when beening discovered and effective treatment methods are unavailable. Therefore, exploring an effective biomarker for early diagnosis and improving the treatment strategies are urgently required.Recently, a novel candidate oncogene, tumor necrosis factor, alpha-induced protein 8(TNFAIP8), has gradually aroused scholars, attention. Tumor necrosis factor, alpha-induced protein 8(TNFAIP8), also known as NDED, GG2-1, SCCS2, SCC-S2, MDC-3.13, is located in 5q23.1 and involved in malignancies of numerous tumors. Recently, the role of TNFAIP8 in the formation of a series of tumors has been verified. These results demonstrated that TNFAIP8 plays a vital role in the progress of the tumor and participates in the regulation of cell prolif eration, invasion, migration, apoptosis and drug resistance among different tumors.However, the expression of TNFAIP8 and its clinical significance in GC has not been well determined. If we can clarify its regulation mechanism and find the corresponding point to intervene, we may be able to find new effective treatment for gastric cancer and improve its prognosis. It will have a very important clinical significance and application prospectsPart ⅠThe expression of TNFAIP8 in human gastric cancerObjective:To confirm the expression of TNFAIP8 in human gastric cancer and explore its relationship with clinicopathological features and prognosis.Materials:Patients and samples.1. Fresh samples of gastric cancer and adjacent normal tissues (4 pairs) were collected for protein expression levels detection of TNFAIP8 via Western blot.2. Furthermore, total 86 cases of gastric cancer tissues and adjacent normal tissues (from 1.1.2007 to 7.1.2008) were obtained from the the Department of Pathology of Shandong Provincial Hospital, which were suffer radical operation therapy, according to the National Comprehensive Cancer Network (NCCN) Practice Guidelines. All samples had not received any preoperative treatment, including chemotherapy and radiotherapy. Gastric cancer tissues were collected from patients and healthy controls at the Provincial Hospital Affiliated to Shandong University, after obtaining the subjects’informed consent and with institutional review board approval of the hospital. All patients obtained a confirmed diagnosis of gastric carcinoma after resection.3.GES-1, MKN-28, SGC-7901, MGC-803 cells were preserved in our laboratory. The human gastric cancer cell line NCI-N87 was provided by the institution of digestive surgery of Ruijin hospital affiliated to Shanghai Jiao Tong University.Methods:1. Immunohistochemical (IHC) staining of tissues and Immunofluorescence.For immunohistochemical staining, tumour specimens were embedded in paraffin and 4-μm thick sections were obtained from Department of Pathology of our hospital. Briefly, the sections were dewaxed according to SABC manufacturer’s instructions. Hydrogen peroxide (0.3%) and blocking serum were implemented to block endogenous non-specific substances. After each blocking, tissues were washed with PBS 5 minutes for three times. Tissues were then incubated at 4℃ overnight with TNFAIP8 rabbit polyclonal antibody (1:100 dilution; Abcam64988, USA). After washing with PBS, tissues were incubated with secondary antibody(Zhong Shan Golden Bridge Biological Technology Inc. Beijing, China) for 30 minutes at 37℃. Then tissues were stained with DAB and hematoxylin. The experiment was repeated for three times. And immunofluorescence technique was also applied to confirm the location of TNFAIP8 protein following the familiar instruction.2. Immunohistochemical staining evaluation.The stained tissues were evaluated by two pathologists who blinded to the exact condition of the patients and the scores were depended on staining intensity and proportion as previously described (12). Based on the intensity, the degree was categorized as follows:O(negative),1 (weak),2 (moderate), and 3 (strong). And the proportion of TNFAIP8 expression staining was scored on a scale of O(absent),1(<25%),2(26%-50%),3 (51%-75%), and 4(>75%). The final result was accumulated the intensity and proportion scores. IHC scores of less than 4 points were determined to low expression and more than 4 points was considered high expression.3. Cell culture.GES-1, MKN-28, SGC-7901, MGC-803 cells were preserved in our laboratory. The human gastric cancer cell line NCI-N87 was provided by the institution of digestive surgery of Ruijin hospital affiliated to Shanghai Jiao Tong University. The cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum(FBS), penicillin (100 U/mL), and streptomycin (100 mg/L) in a humidified atmosphere containing 5% CO2 maintained at 37℃.4. Real-time quantitative PCR (qPCR).Total cellular RNA was isolated from cells utilizing the TRIzol Reagent (Invitrogen, Carlsbad, CA) depending on the manufacturer’s protocol. The RNA was reverse transcribed and the resulting cDNA samples were amplified by real-time PCR (The LightCycler 480 Real-Time PCR System, Roche, China) using gene-specific primers(TaKaRa Biotechnology(Dalian)Co., Ltd.). The sequences of the primer pairs are as follows: TNFAIP8 forward:5’-TTCCATCAGGTGGATTATACCTTTG-3’. TNFAIP8 reverse:5’-AGGTGGCGCTGAATGATTTG-3’. mRNA levels were normalized to GAPDH.5. Western blotting analysis.Briefly, tissues and cells were washed, lysed and harvested. Protein concentration in the resulting lysate was evaluated using the bicinchoninic acid protein assay Kit (Pierce Biotechnology, Rockford, IL). Appropriate amounts of protein were separated by electrophoresis in 12% Tris-glycine polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked and then incubated overnight at 4℃ with the rabbit anti-human TNFAIP8 primary antibody (1:500 dilution; Abcam64988, USA). Then next day membranes were washed three times with TBST for 10 min and incubated with the corresponding secondary antibody for 1h. After washing three times for 10 min, bound secondary antibody was detected using an enhanced chemiluminescence (ECL) system (Pierce Biotechnology Inc., Rockford, IL, USA). Protein levels were normalized to β-actin.Results:1. The tissue specimens by immunohistochemistry and immunofluorescence assay showed the expression of TNFAIP8 in gastric cancer cell lines and gastric tissus and the expression was mainly localized in the cytoplasm.2. Immunohistochemical staining scores show that in tumor tissue, TNFAIP8 gene expression in T3 and T4 stage(n=56) was significantly higher than in T1 and T2 stage(n=30) (p=0.0013), whereas the expression in the adjacent normal tissues was no significant difference (p=0.6831).3. Western blot analysis of TNFAIP8 in gastric cancer tissues and cell lines shows that expression of TNFAIP8 in gastric cancer cell lines was compared to normal gastric mucosal cells. (P<0.05, Student’s t-test).Conclusion:1. TNFAIP8 is expressed in gastric cancer cell lines and gastric tissus and is considered to be a cytosolic protein.2. Research shows there is a significant correlation between TNFAIP8 upregulation expression and clinicopathological features and poor prognosis.3. The expression level of TNFAIP8 in gastric tissus is higher than normal tissus.Part ⅡThe effect of TNFAIP8 on regulation of cell proliferation, invasion andmigrationObjective:In the first step of the experiment, we have confirmed TNFAIP8 highly expressed in gastric tissues and cells, and then, our research group will knockout TNFAIP8 and then detect potential mechanism of proliferation, invasion and migration.Methods:1. Sequence design and vector construct.siRNA Treatment:Small interference RNA to TNFAIP8 was synthesized by Shanghai Genechem Co,. Ltd. The DNA target sequence for siRNA-TNFAIP8 (5’-CCACCTTAATAGACGACACAA-3’)was designed based on the core sequence of human TNFAIP8 cDNA. The cells who were successfully transfected with the siRNA according to the manufacturer’s instructions and were observed in the microscope.2. Lentivirus transfection.For transfection, SGC-7901 and MKN-28 gastric cancer cells were pre-cultured in 96-well plates at a density of 5×103 cells per well. Cells were infected with the lentiviral vectors at different MOIS (multiplicity of infection, or infectious units ratio), when they were about 50%-60% confluent. Transfection efficiency was observed with an inverted fluorescence microscope (DP72, Olympus, Japan) after 72 hours.3. Western blotting analysis.Same method with the first part of the experiment.4. Colony formation assay.Gastric cancer cells were digested into single suspension cells and then 1×103 cells were plated in 60mm plates with 4ml complete culture medium. The plates were cultured at 37℃ in 5% CO2 for ten days. Colonies including at least 50 cells is considered to be statistically significance. The results are listed as the mean ± standard deviation from five randomly selected fields.5. CCK-8 assay.Gastric cancer cells (8×102 cells) were incubated in 96-well plates with 100ul 10% fetal bovine serum medium. Cells were continuously incubated for 24,48, 72,96, and 120 hours at 37℃ in 5% CO2 and cell number was estimated using the cell counting kit-8 (CCK-8) (Dojindo C0038, Japan). Briefly,10 uL CCK-8 was added to each well, after 1 hour of incubation, absorbance at 450 nm was measured to calculated the number of cells. Each type were repeated for six independent times.6. Invasion and migration assay.For invasion assays,3×105 cells with 200 ul medium were plated in the upper chamber which was divided with the lower chamber by 50 ul Matrigel-coated membranes (24-well insert,8-μm pore size; Corning, NY, USA). For the migration assay, the membranes were not coated with Matrigel and the culture condition was as same as the invasion assay. Finally, for the two assay, the membranes were cut down and stained with hematoxylin and photographed.7. Statistical analysis.Statistical analysis was determined using SPSS18.0 software(SPSS inc., USA). Protein and mRNA expression of TNFAIP8 in human gastric cancer cell lines and tissues were expressed as means ± SD with at least three independent experiments. Chi-square and Fisher’s exact tests were used for categorical variables. P<0.05 was considered to be statistically significant.Results:1. Western blot analysis indicated that TNFAIP8 protein expression treated with siRNA was significantly suppressed compared to negative control. QRT-PCR showed that treatment with siRNA markedly decreases TNFAIP8 mRNA levels in gastric cancer cells.2. CCK-8 assay showed that decreased TNFAIP8 expression levels inhibited proliferation in SGC-7901 and MKN-28 cell lines. Colony formation assay illuminated that cells were decreased significantly in the groups with TNFAIP8 siRNA treatment compared with the negtive controls. Transwell assay elucidated that cells treated with TNFAIP8 siRNA have poor invasive and migratory capacities than negative controls. Single asterisk indicates p<0.05.3. Invasion and migration assay showed that decreased TNFAIP8 expression levels inhibited cell invasion and migration.Conclusion:1. Western blot and QRT-PCR analysis indicated that TNFAIP8 protein expression and mRNA levels were markedly decreases in gastric cancer cells treated with siRNA.2. CCK-8 assay and colony formation assay showed that decreased TNFAIP8 expression levels inhibited proliferation in gastric cancer cells.3. Invasion and migration assay showed that decreased TNFAIP8 expression levels inhibited cell invasion and migration.