| BackgroundGastric cancer is one of the most common malignancy worldwide, and more than 700000 people die of gastric cancer in 2014 makes this disease the third most common cause of cancer death globally. Despite the development and identifcation of novel anticancer agents and treatment methods were applied for present clinical therapy, almost 50% of gastric cancer patients suffer from recurrences disease and distant metastases within 5 years after the surgical treatment. The main cause of the recurrence remains unclear.IL-17 is a proinflammatory cytokine mainly secrete produced by Th17 cell subsets and neutrophils(proinflammatory cytokine), which was composed by a relative molecular mass of 155 amino acids(15-22) × 103 of the homodimer and N terminal signal peptide. IL-17 cytokine family have at least six members, including IL-17 A, IL-17 B, IL-17 C, IL-17 D, IL-17E(also called IL-25) and IL-17 F, IL-17 A, also known as IL-17 or CTLA-8, was recognised as the iconic cytokine family(hallmark cytokine). Chronic inflammatory disorders such as inflammatory bowel disease(inflammatory bowel diseases), Helicobacter pylori infection, hepatitis B virus infection and prostatitis can induce some unknown pathogen tumors, named as inflammation-associated cancer, and in these tumors there was increased expression of IL-17 found. We also explored the associations between expression of IL-17 in gastric cancer and the depth of invasion, lymph node metastasis and TNM stage in previous studies, then we discovered that patients with high expression IL-17 in gastric cancer tissue combined with poor prognosis. However, the function of IL-17 for invasion and metastasis gastric cancer cells and its mechanism were still uncertain.Epithelial-mesenchymal transition(EMT) is the phenomenon that epithelial cells lose their cell characteristics and gain migration and invation propreties to become mesenchymal cells. This process changes the molecular level occurs and cell morphology related to changes. A number of recent studies have confirmed that epithelial-mesenchymal transformation was closely related to tumor invasion and metastasis. Tumor cells of epithelial origin interstitial qualitative transformation decreased intercellular adhesion, cell and matrix adhesion diminished capacity, exercise capacity, these features changes invasion and migration are provided conditions, playing important roles in the invasion and metastasis of malignant tumor cellsTumor microenvironment is a complex environment composed by the tumor cells, stromal cells, immune cells, and a number of cytokines, previous literatures reported that inflammatory cytokines and inflammatory mediators based on inflammatory tumor microenvironment plays an important role in invasion and metastasis factors. on the one hand, tumor cells are able to activate the immune system, a number of inflammatory cytokines play a role in tumor suppression, on the other hand, inflammatory factors alsocan promote angiogenesis, activation of certain signaling pathways, promoting tumor cells invasion and metastasis.Based on remarkable function of IL-17 in the invasion and metastasis of gastric cancer, we hypothesized that IL-17 may promote epithelial-mesenchymal transformation of gastric cancer cells and induce indirect effects of gastric cancer invasion and metastasis.In the present study, we used in vitro cell models to study the effect of IL-17 in epithelial-mesenchymal transition in gastric cells by PCR, WB,, and wound healing assay, cell invasion assay and other means.MethodsFirst, effects of different concentrations of IL-17 on the morphological changes of gastric cancer cellsTake the cell in SGC-7901 cells and MGC-803 cells to used as the experimental cells. The final concentration of 0ng/m L,1 ng/mL, 10 ng/mL, 100 ng/mL, 1ug/m L IL-17. cells were cultured for 48 hours. The morphological changes of the cells were observed under the action of IL-17.Second, effect of IL-17 on the migration of gastric cancer cellsThe migration of gastric cancer cells was detected by conventional scratch test method. The experimental group was added to 100 ng/mL of IL-17 for 48 hours. The control group was added with equal amount of PBS, and the scratch width was recorded. cells were cultured for 24 hours.Three, effect of IL-17 on the invasion ability of gastric cancer cellsInvasion ability, select the cell suspension via Transwell chamber assay method to detect gastric cancer cells, the experimental group with 100 ng/mL concentrations of IL-17, control group were treated with equal volume of PBS and culture for 48 hours. Cells were seeded onto Transwell chamber, chamber containing fetal bovine serum, then put into the incubator in cultured for 24 hours. Remove the Transwell chamber, after the wash, fixation, staining and counting.Four, effect of IL-17 on the expression of Vimentin, N-cadherin and E-cadherin RNA in gastric cancer cellsPCR m RNA was used to detect the expression of ng/mL in gastric cancer cells, and the control group was added to 100ng/m L of IL-17, and the control group was added to the PBS, which was cultured for 48 hours. The total protein was extracted, and the expression of E-cadherin, N-cadherin and Vimentin RNA in real-time RNA.Fifth, effect of IL-17 on the levels of Vimentin, N-cadherin and E-cadherin proteins in gastric cancer cellsWestern Bolt assay was used to detect the expression of gastric cancer cells, and the experimental group was added to 100 ng/mL of IL-17 concentration, and the control group was added to the PBS, which was cultured for 48 hours. The total protein was extracted, and the expression of E-cadherin, N-cadherin and Vimentin in protein level was determined.Result:1 Effects of different concentrations of IL-17 on the morphological changes of gastric cancer cellsThe results showed that: in the cell morphology of gastric cancer cells, we found that the change of cell morphology after IL-17 treatment, the concentration was dependent. IL-17 100ng/mL treatment, 24 hours on the cell morphology, we observe the cell adhesion was reduced, the cell is changed into spindle shaped cells, some cells become round, and even suspended.2.Effect of IL-17 on the migration of gastric cancer cellsThe results showed that the scratch width of SGC-7901 gastric cancer cells was 0.41±0.09 mm, Control group scratch width 0.71±0.09 mm,(P=0.0188). There were significant differences between the two groups, the experimental group was significantly better than the control group.The results showed that the scratch width of MGC-803 gastric cancer cells was the scratch width was 0.40±0.15 mm, and the control group was 0.76±0.13 mm,(P=0.0364). There were significant differences between the two groups, the experimental group was significantly better than the control group.2.Effect of IL-17 on the invasion ability of gastric cancer cellsThe results showed that the number of the Gastric cancer of SGC-7901 cells in the experimental group was 170 ± 15, the number of the control group was 131 ± 14,(P=0.0331). There were significant differences between the two groups, and the invasion ability of the experimental group was significantly higher than that of the control group.The number of the Gastric cancer of MGC-803 cells in the experimental group was 159±28, the number of the control group was 94±18,(P=0.0297). There were significant differences between the two groups, and the invasion ability of the experimental group was significantly higher than that of the control group.3.Effect of IL-17 on the expression of E-cadherin,Vimentin and N-cadherin RNA in gastric cancer cellsThe results showed that the relative expression of m RNA E-cadherin in gastric cancer of SGC-7901 cells was 0.81±0.14,the control group was 0.97±0.16(P=0.0222), the relative expression of m RNA N-cadherin was 1.16±0.12, the control group was 0.71±0.13,(P=0.014), the relative expression of mRNA Vimentin was 1.51±0.22, the control group was 0.81±0.13,(P=0.0096). There were significant differences between the two groups, the expression of E-cadherin in the experimental group was significantly lower than that in the control group, and the expression of Vimentin and N-cadherin was significantly higher than that in the control group.The relative expression of mRNA E-cadherin in gastric cancer of MGC-803 cells was 0.45±0.13,the control group was 1.06±0.23(P=0.0189), the relative expression of mRNA N-cadherin was 1.36±0.20, the control group was 0.89±0.13,(P=0.0289), the relative expression of m RNA Vimentin was 1.51±0.22, the control group was 1.23±0.41,(P=0.0431)There were significant differences between the two groups, the expression of E-cadherin in the experimental group was significantly lower than that in the control group, and the expression of Vimentin and N-cadherin was significantly higher than that in the control group.4.Effect of IL-17 on the levels of Vimentin, N-cadherin and E-cadherin proteins in gastric cancer cellsThe results showed that the relative expression of protein E-cadherin in gastric cancer of SGC-7901 cells was 1.08±0.37,the control group was 1.82±0.22(P=0.0418), the relative expression of protein N-cadherin was 1.42±0.22, the control group was 0.84±0.16,(P=0.0209), the relative expression of protein Vimentin was 1.65±0.27, the control group was 0.95±0.10,(P=0.0146). There were significant differences between the two groups, the expression of E-cadherin in the experimental group was significantly lower than that in the control group, and the expression of Vimentin and N-cadherin was significantly higher than that in the control group.The relative expression of protein E-cadherin in gastric cancer of MGC-803 cells was 0.86±0.17,the control group was 1.56±0.29(P=0.0238), the relative expression of protein N-cadherin was 1.41±0.35, the control group was 0.77±0.15,(P=0.0441), the relative expression of protein Vimentin was 1.65±0.27, the control group was 0.95±0.10,(P=0.0227)。There were significant differences between the two groups, the expression of E-cadherin in the experimental group was significantly lower than that in the control group, and the expression of Vimentin and N-cadherin was significantly higher than that in the control group.Conclusion:1.IL-17 can significantly change the morphology of gastric cancer cells, and significantly enhance the invasion and metastasis of gastric cancer cells.2.IL-17 can significantly reduce the level of expression of E-cadherin epithelial markers in gastric cancer cells, and significantly enhance the expression level N-cadherin and Vimentin of stromal markers, which can increase the conversion of gastric epithelial cells.3 Epithelial mesenchymal transition is one of the important mechanisms of IL-17 promoting the invasion and metastasis of gastric cancer cells. |
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