Expression And Functional Study Of Insulin-like Growth Factor Binding Protein 7 In Human Gastric Carcinoma

Gastric cancer is one of the most common malignant tumor of the world and is the fourth morbidity and the second mortality in overall cancers worldwide, has been the high gastric cancer occurrence country for a long time. Although in recent years, the morbidity and mortality of gastric cancer worldwide trendcy have been declining, there are still 900,000 new cases and 700000 patients died of stomach cancer each year around the world. In China, there are about 300,000 new cases of stomach cancer. Although with the development of science, surgery, radiotherapy, a series of chemotherapy drugs and targeted drugs are widely used, the effectiveness for the advanced gastric cancer, the prognosis of metastatic gastric cancer remains is poor. Therefore, seeking for molecular markers in early diagnosis and the new potential therapeutic targets of gastric cancer are particularly important.Insulin-like growth factor binding protein 7 (IGFBP7), belongs to IGFBPs family. It locates in 4 q12, transcription stretches and has the size approximately 1100 KB with five exons. In 1993, Murphy et al. for the first time found that the is gene expression is significantly higher in normal meninges cell lines than in the meninges tumor cell lines, and named as MAC25, IGFBP7 gene is called a tumour adhesion factor (tumor derived adhesion factor, TAF the prostate gland ring element stimulating factor (porstacyclin-stimulating facotr, PSF), IGFBP associated protein 1 (IGFBP-related proteinl). The study found that IGFBP7 was a secreted protein, which expressed in multiple tissues in normal human body, including respiratory, digestive, urinary and reproductive, nervous, immune and endocrine systems, IGFBP7 are positive expressed in almost all endothelial cells except the brain and spinal cord. The specificity of the IGFBP7 expression level and tissue location implies the different biological roles of IGFBP7.In recent years, numerous researches reported the effects of IGFBP7 in development, but the results were inconsistent. Its espression is low expressed in column adenocarcinoma, breast cancer, colon cancer, lung cancer, liver cancer, meningioma, melanoma, esophageal adenocarcinoma, but high in pancreatic cancer, leukemia, esophageal squamous carcinoma. Both low and high expressions were found in glioma. A number of studies showed that IGFBP7 is associated with the development of tumor gene, while little is known about the effect of IGFBP7 on gastric cancer, either its influences on gastric malignant biological phenotype or its suppressing or promoting effects on gastric cancer. Further investigation is necesasry.The goal of this study is to investigate the expression of IGFBP7 in human gastric cancer tissue, the biology role of this gene and the relevant molecular mechanisms, and its role on the growth of nude mice. At first, Real-time PCR, Western blot, IHC were employed to analyze the IGFBP7 expression in gastric cancer tissue and and the association between its expression with the tumor clinical pathology. The reault showed that the expression of IGFBP7 was low in gastric cancer tissue and the patients who had low expressed of IGFBP7 showed a poor post-surgery prognosis. Then we transfected the IGFBP7 to the gastric cancer cells SGC-7901, which had no endogenous expression of IGFBP7 and transfected si-IGFBP7 to the gastric cancer cells MKN-28, which had the expression of IGFBP7. Through the assays of Real-time PCR, Western blot, MTT, flow cytometry, cell scratches, Transwell chambers, it was found that IGFBP7 over-expression in tumor cells could inhibit the prolifertion, mignant, and invasion, and promote apoptosis of gastric cancer cells, and up regulate E-cadherin (epithelial marker) and Bax (apoptosis marker), but down regulate the expressions of CyclinDl, CDK4 (the regulating factors of cellular period) MMP2 (mesenchyme marker), and Bcl2. The similar results were found in gastric cancer cells MKN-28. The low expression of IGFBP7 could promote cell growth, decrease the cell number at G1 stage, promove the migrationg of cancer cells, and inhibit apoptosis. Finally, we built a nude mouse model and made subcutaneous injection of single cell suspension SGC-7901 cells into different groups of nude mouse to estabolish the tumor bearing nude mice for the purposes of investigation the influence of IGFBP7 on characteristics of gastric cancer cell in vivo. The result showed that IGFBP7 could inhibit the growth of gastric cancer in nude mouse.Objectives1. To study the expression of IGFBP7 in human gastric cancer tissue, the association between its expression level and the clinical pathological features;2. To study the biological role of IGFBP7 in malignant gastric cancer cell and the possible molecular mechanism in vitro;3. To study the effects of IGFBP7 on the growth of the gastric cancer xenografts.MethodsClinical trials1. The specimens were collected from fresh tissue adjacent to carcinoma and gastric cancer tissue in the first affiliated hospital of Zhengzhou university in the department of general surgery after operation and then further precessed to make paraffin embedding blocks.2. The assays of Real-time QT PCR, western blot, and immunohistochemistry staining technology were carried out to detect the levels of mRNA and protein expressions of IGFBP7 in gastric cancer tissue and the corresponding expression of tissue adjacent to carcinoma. The association between GFBP7 expression and clinical pathological was analyzed.The effects of GFBP7 expression on gastric cancer cells1. The expressions of IGFBP7 in various cell lines were retected using Real-time QT PCR, western blot in high differentiation of gastric cancer cell lines MKN-28, moderate differentiation of gastric cancer cell line SGC-7901 and AGS, poorly differentiated stomach cancer cell lines MKN-45 and HGC-27, normal gastric mucosa epithelial cells immortalized the GES-1. The subsequent cell lines were selected and used in the following experiments.2. The two cell lines (SGC-7901 and MKN-28) were separately transfected. The experiments were set as (1) the group of SGC-7901 over-expression:the blank control group, negative control and transfection PcDNA3.1-IGFBP7 group; (2) the groups of MKN-28 knockdown:the blank control group, negative control, and siRNA-IGFBP7 group. After 48 hr of transinfection, the expressions of the IGFBP7 at mRNA and protein levels were neasured through RT-PCR and western blot methods and the expression efficiency was evaluated.3. The influences on cell proliferation and cellulsr cycle of SGC-7901 and MKN-28 cells by expression of IGFBP7 was assessed through MTT experiment and flow cytometry.4. The cells of each group were cultured in corresponding medium without fetal bovine serum (FBS) for 12 h and then scratched with a pipette tip. Wound areas were marked and photographed at 0 h,12 h,24 h and 36 h respectively. The rate of cellular migration was evaluated by both photographing and quantifying the migrated distance of cells moved from the wound edge toward the center using IPP (Image-Pro Plus 6.0) software. Each group cells were incubated for 24 hr. After 24 hours, transwell migration assay and Matrigel invasion assay were performed separately using 24-well Transwell inserts with 8μm pore size (Corning Costar Corp). For Transwell migration assay, the SGC-7901 or MKN-28 cell number of 2×104 from each group were suspended in 100 μl of corresponding culture medium without FBS were loaded into the top chamber of transwell inserted with non-coated membrane. For Matrigel invasion assay, total of 5×104 SGC-7901 or MKN-28 cells were plated in 100μl of serum-free medium in the upper Matrigel-coated chamber instead. In both assays, the bottom chamber contained 600 μl of medium with 20% FBS. Cells were then allowed to migrated or invaded for 12 h at 37 ℃. The cells that migrated or invaded into the bottom chamber were fixed, stained with 1% crystal violet, visualized under phase contrast microscope and photographed.5. The apoptosis of transfected SGC-7901 and MKN-28 cells in all groups were evaluated by flow cytometry.6. Through western blotting assay to explore the effects of IGFBP7 on cellular period, invading, and apopotosis and the possible molecular mechanism.Gastric cancer model of nude mice1. The stable IGFBP7 expression SGC-7901 strains were established and screened first.2. The nude mice were randomly grouped into testing group (stable expression IGFBP7 SGC-7901) and control group (SGC-7901). The tumor cells in the number of 2×107 (in the volume of 200μl) were injected into every BALB/c nude mice subcutaneously.3. The tumor size and body weight of the nude mice were measured every four days. According to the measured data, the growth curve of the tumor was drawn. On the day of 20th, the mice were euthanized and the tumor tissue was dissected. The protein expression of IGFBP7 in tumor cells was evaluated through HE and IHC stain and flowed by microscope analysis.Statistical analysisData were processed and analyzed using SPSS 17.0 software for statistical analysis. The data were expressed as mean±standard deviation (x±s), χ2 test, LSD(Least-significant difference) test, Tamhane’s T2 test and Student’s t-test were applied. The significant level was set at a=0.05.Results1. The results of qRT-PCR, Western blot and IHC method showed that IGFBP7 mRNA and protein expression in gastric cancer tissue were significantly lower than the corresponding normal gastric mucosa tissue adjacent to carcinoma (P<0.01). Low expression of IGFBP7 were related to the the T staging (P=0.037) and pathological grade (P=0.021), Kaplan Meier survival curve showed that patients with negative tumor cell expression of IGFBP7 had shorter postoperative survival length.2. The expression of IGFBP7 was lower in MKN-28 cell lines than that in GES1 cell lines, while the expression of IGFBP7 was very low or even non-detectable in the cell lines of SGC-7901, AGS, MKN-45, and HGC-27, which is similar with the expression patern in gastric tumor cells.3. IGFBP7 expression level was significantly increased in the PcDNA31.-IGFBP7 transfected SGC-7901 cells, but decreased in the siRNA-IGFBP7 transfected MKN-28 cells than the controls.4. The MTT experiments showed that the growth was significantly slower in the PcDNA3.1-IGFBP7 transfected SGC-7901 cells than that in the control cells (P< 0.05). The growth and proliferation was significantly faster in consiRNA-IGFBP7 transfected MKN-28 cell than that in siRNA-IGFBP7 tansfected MKN-28 cells (P<7. The effect IGFBP7 on apoptosis showed that the apoptosis was significantly increased in PcDNA3.1-IGFBP7 transfected SGC-7901 cells, while decreased in siRNA-IGFBP7 tansfected MKN-28 cells.8. Western blotting results showed that the up-regulated expression of IGFBP7 could inhibit the expressions of Cyclin D1, CDK4, MMP2, Bcl2, which further decreased the expressions of E-cadherin and Bax, but down regulated expressions of IGFBP7 could promote the expression of Cyclin D1, CDK4, MMP2, Bcl2, which further decreased the expressions of E-cadherin and Bax.9. Nude mouse transplantation tumor experiments showed that the mice body mass was not significantly different between the the test and control groups (P> 0.05). The tumor size with the transfection of PcDNA3.1-IGFBP7 was significantly smaller than the control (P< 0.05). The inhibit rate of tumor growth was about 25.53%. IHC testing showed that IGFBP7 protein expression levels were significantly higher in PcDNA3.1-IGFBP7 transfection mice than the controls (P< 0.05).Conclusion1. The expression of IGFBP7 is significantly lower in gastric cancer tissue than the corresponding tissue adjacent to carcinoma and the low expression implys the poor prognosis of surgery;2. Up-regulated expression of IGFBP7 can inhibit cellular proliferation, the migration and invasion, keep the tumor cell at G1 stage, and promote cell apoptosis in gastric cancer cell. Down-regulated expression of IGFBP7 can promote cellular proliferation, the migration and invasion, decrease the cellular number at G1 stage, and inhibit cell apoptosis in gastric cancer cell.3. PcDNA3.1-IGFBP7 could inhibit the growth of xenograft of human gastric cancer in nuke mice, which implys that IGFBP7 gene is involved in inhibition of gastric cancer cell proliferation.4. The expression of IGFBP7 is low in gastric cancer. It is confirmed in both in vivo and in vitro experiments that IGFBP7 plays important roles in gastric cancer development. The study result might provide theoretical foundation for the clinical application of IGFBP7 agonist.