| BackgroundGastric carcinoma (GC) is the fourth most common malignant tumor in the world. In China, the incidence of GC ranks second and the mortality ranks third among all malignant tumors. Although the mortality of GC has declined in recent years primarily due to improvements in radical resections for gastric cancer and combination chemotherapy, the prognosis of GC remains poor because of the high incidence of lymph node metastasis and vascular invasion. Therefore, the exploration of GC initiation and progression mechanisms may improve early prevention and treatment efficacy.The close connection between inflammatory immune response and tumorigenesis and the underlying mechanism have been extensively investigated. The inflammatory immune response relies on cytokines and immune cells to recognize tumor antigens and eliminate tumor cells, which is addressed within the frame of the "tumor immunosurveillance". On the other hand, the inflammatory immune response can also result in induction of "mutagenic" enzymes,epigenetic modification and gene mutation which contribute to tumor promotion, progression and metastatic spread (tumor escape).Helicobacter pylori(H. pylori) is recognized as carcinogen of GC by the World Health Organization. Chronic Helicobacter pylori infection can induce chronic atrophic gastritis, thereby causing dysplasia and gastric cancer. Therefore, the inflammation responses play critical roles at tumor-initiating stage. Cytokines as a regulator of the inflammatory response may play an important role in tumor promotion and progression. Interleukin(IL)-6 is one of the best-characterized pro-tumorigenic cytokines in tumor microenvironment. IL6 can directly promote cancer cell proliferation, invasion and metastasis and the SNPs in its promoter region are closely associated with gastrointestinal cancer(esophageal cancer, liver cancer, colon cancer) susceptibility. Tocilizumab, one of IL6 neutralizing antibodies, is in Phase Ⅱ Clinical Trials for targeted cancer therapy and adjuvant therapy. IL-11 is a member of the IL-6 family of cytokines, which mediate signaling via a common signal-transducing gp130 component and a cytokine-specific subunit. IL-11 was originally cloned as a soluble mediator in the supernatant of cultured fibroblasts that could stimulate the growth of IL6-dependent plasmacytoma cells and associated IgG production. IL11 was later found to exhibit a wide variety of biological effects in neural cells as well as in the hematopoietic and immune systems. For example, IL11 can stimulate erythropoiesis and thrombopoietic activity. IL11 also regulates polarization of T-cells and macrophages, promotes maturation of bone resorbing osteoclasts and associated net bone loss, retains tissue "stem cell" phenotypes. Over the past decade IL11 was found to play a more important role in gastric cancer promotion and progression than IL6.1) Homozygous gp130757F/F knock-in mice, where this critical tyrosine is replaced by a phenylalanine, develop gastric hyperplasia and tumors in antrum. The negative regulator SOCS3 can not bind to the the replaced phenylalanine and can not terminate GP130-STAT3 signal. Therefore, the lack of negative signal regulation in gpl30-757F/F knock-in mice leads to hyper-activation of STAT3 signaling and is solely dependent on IL-11. Gp130-757F/F mice showed a more than 30-fold up-regulation of IL-11 mRNA within the gastric tumors. Gp130-757F/F mice crossed onto an IL-6 deficient background showed no reduction in tumor burden, whereas gp130-757F/F:IL-11Ra deficient mice developed no gastric tumors. 2) IL-11 protein was localized predominantly to parietal cells in mouse and human stomach. Intraperitoneal application of exogenous IL-11 in wild mice resulted in fundic parietal and chief cell loss, hyperplasia, mucous cell metaplasia and inflammation. Coincident with cellular changes were an increased gastric pH, altered parietal cell ultrastructure and altered gene expression, particularly genes involved in immune response and ion transport which could result in compromised acid secretion. IL-11 is a parietal cell cytokine that leads to chronic atrophic gastritis and is involved in gastric tumorigenesis.3)Exogenous IL-11 increases gastric cancer cell motility in vitro. But there are still some questions to be addressed in IL11-centered gastric cancer promotion and progression:Is the expression of IL11 in chronic non-atrophy gastritis different from that in precancerous lesions(atrophy gastritis and intraepithelial neoplasia)? What is the relationship between the expression of IL11/IL11RA in gastric carcinoma and the overall survival rate of patients? What is the molecular mechanisms and signaling pathways by which IL11 promotes cancer cell migration and invasion? Is the SNPs in IL11 /IL11RA gene closely associated with the risk of gastric cancer development? So we adopt histology methodology, cell biology methodology and molecular epidemiology methodology to elaborate these issues.Methods1. The expression of IL11 and IL11RA was examined by immunohistochemistry in 64 mucosal specimens including 16 cases of chronic non-atrophy gastritis,16 cases of chronic atrophy gastritis,16 cases of low-grade intraepithelial neoplasia and 16 cases of high-grade intraepithelial neoplasia. The status of Helicobacter pylori infection was also recorded.The association between the status of Helicobacter pylori infection and the expression of IL11 and IL11RA were further determined.2. The expression of IL11and IL11RA was investigated by immunohistochemistry in 167 cases of resected gastric carcinoma tissues. The relationship between IL11/ IL11RA expression and gastric carcinoma differentiation, lymph node metastasis,local invasion and overall survival time was carefully analysised.3. The expression of IL11 and IL11RA in normal gastric epithelial cell line and several gastric cancer cell lines was detected by Western blotting. The gastric cancer cell lines stably expressing IL11RA were selected.4. Effect of rhILll on the proliferation of MGC803 and SGC7901 cell lines was assayed by Cell Counting Kit-8 assay.5. Effect of rhILll on the migration of MGC803 and SGC7901 cell lines was evaluated using a scratch migration assay and a Transwell migration study. The STAT3 inhibitor S31-201 or PI3K inhibitor LY294002 was added to the low chamber containing rhILll and the effect of these inhibitors on the migration of MGC803 and SGC7901 cell lines were assayed.6. Effect of rhIL11 on the invasion of MGC803 cell line was evaluated using a matrigel invasion assay. The STAT3 inhibitor S31-201 or PI3K inhibitor LY294002 was added to the low chamber containing rhIL11 and the effect of these inhibitors on the invasion of MGC803 cell was also assayed.7. Activation of signal transduction elements(STAT3, p-STAT3, ERK, p-ERK,AKT, p-AKT) in response to rhIL11 stimulation was assayed by Western blotting.8. Effect of rhILll on the expression of COX2, VEGF, HIF-la in MGC803 cell line was evaluated by Q-RT-PCR at four different time points(hours 0,1,6,24).9. Effect of rhIL11 on the expression of ICAM-1, MMP2, MMP9, E-cadherin, Vimentin in MGC803 cell line was evaluated by Western blotting at five different time points(hours 0,12,24,36,72).10. MGC803 cell was maintained in serum-free medium(DMEM) supplemented with rh IL11 (100ng/ml)or not in five days. The cell growth pattern and cell morphology were observed and compared between two groups under a microscope.11. Effect of rhILll on the expression of E-cadherin, Vimentin in MGC803 cell line was evaluated by immunofluorescence after 36 hours stimulation.12. Western blotting was employed to determind whether STAT3 or AKT depletion may affect expression of E-cadherin and Vimentin regulated by rhILll in MGC803 cell line.13. A total of 108 cases of gastric cancer patients and 367 cases of normal control were enrolled. Genomic DNA was isolated and purified from the whole blood. Polymerase chain reactions were performed using specific primers for the IL11 and IL11RA SNPs(rs1126757,rs4252546,rs1061758,rs2812357). SNP genotyping was conducted by direct sequencing.14. Statistical software SPSS 13.0 (Statistical Package for the Social Sciences, SPSS, Chicago, Illinois, USA) was used for statistical analyses. The association between IL11/IL11RA expression and GC clinicopathological features was analyzed using a χ2 test. The Kaplan-Meier method was used to analyze cumulative survival rate. Results obtained from qPCR, migration, invasion, cell growth experiments were expressed as the means±SD. If equal variances were assumed, two-tailed Student’s t test or one way ANOVA were performed; if equal variances were not assumed, Dunnett’s T3 test was used. The Pearson’s χ2. test was used to compare the distribution of genotypes in four SNPs between cases and controls. Unconditional logistic regression model was used to evaluate the association between genotypes and risk of GC and odds ratio (OR) and 95% confidence interval (95%CI) adjusted by age and sex were calculated, p< 0.05 was considered significant.Results1. Expression of IL11 was detected in chronic non-atrophy gastritis and gastric precancerous lesions (chronic atrophy gastritis, low-grade intraepithelial neoplasia and high-grade intraepithelial neoplasia) and mainly localized in the cytoplasm. No significant difference in IL11 expression was found between the groups (p=0.475). Expression of IL11RA was also detected in chronic non-atrophy gastritis and gastric precancerous lesions and mainly localized in the cytoplasm. No significant differences in IL11RA expression were observed between chronic non-atrophy gastritis group and chronic atrophy gastritis group (p=0.073) No significant differences in IL11RA expression were observed between low-grade intraepithelial neoplasia group and high-grade intraepithelial neoplasia group (p=0.075). But the expression of IL11RA in low-grade intraepithelial neoplasia group and high-grade intraepithelial neoplasia group was higher than that in chronic non-atrophy gastritis group and chronic atrophy gastritis group(p<0.01, p<0.01 and p=0.005, p<0.01, respectively).2. Expression of IL11 in chronic non-atrophy gastritis and gastric precancerous lesions was closely associated with the status of H. pylori infection. The percentage of high-expression of IL11 in H. pylori infection positive group was higher that in H. pylori infection negative group(66.7%vs 37.8%,p=0.023). No significant differences in IL11RA high-expression proportion were observed between H. pylori infection positive group and H. pylori infection negative group(59.3%vs 35.1%, p=0.056).3. Expression of IL11 in GC was not associated with tumor differentiation (p=0.264).But it was correlated with tumor infiltration and lymph node metastasis(p<0.05 and p<0.05, respectively). Gastric cancer with higher IL11 expression showed deeper infiltration and more lymph node metastasis. Expression of IL11RA in GC was not associated with tumor differentiation, infiltration and lymph node metastasis(p=0.860, p=0.870, p=0.416, respectively).4. Expression of IL11 in GC was closely associated with 5-year survival rate. Patients with high IL11 expression had significantly shorter over survival than patients with low IL11 expression (66% VS 28.2%, p<0.05). Expression of IL11RA in GC was not correlated with 5-year survival rate. No significant differences in over survival were observed between patients with high IL11 expression and patients with low IL11 expression(47.5% VS 39.1%, p=0.424).5. The normal gastric epithelial cell line GES did not expressed IL11RA The gastric cancer cell line MGC803 expressed IL11RA at the highest level. The gastric cancer cell lines SGC7901 and AGS expressed IL11RA at the moderate level. The gastric cancer cell line BGC823 did not expressed IL11RA.6. MGC803 and SGC7901 cells were stimulated by 10%FBS,0% FBS or rhILll (100ng/ml) at four times(hour 24,48,72 and 96). There were no differences in the proliferation of MGC803 cell line stimulated by 0% FBS or rhILll at hour 24,48,72 and 96 (p=0.77, p=0.096, p=0.849, p=0.115, respectively). The effect on the proliferation of MGC803 cell line stimulated by 10%FBS was more obvious than that on the proliferation of MGC803 cell line stimulated by 0%FBS or rhILll (p<0.05). There were no differences in the proliferation of SGC7901 cell line stimulated by 0% FBS and rhILll at hour 24,48,72 and 96 (p=0.295, p=0.058, p=0.083, p=0.727, respectively). The effect on the proliferation of SGC7901 cell line stimulated by 10%FBS was more obvious than that on the proliferation of SGC7901 cell line stimulated by 0%FBS or rhIL11(p<0.05).7. MGC803 and SGC7901 cells were treated with 10%FBS,0% FBS or rhILll (100ng/ml) and a scratch migration assay was used to assess the migration. After 24h incubation, the cells can be seen migrating into the wound with a decreased distance between the edges. After 48h incubation, the wounds were almostly covered by migratory cells in 10%FBS group and 0%FBS+IL11 group, which was more obvious than that in 0%FBS group for MGC803 cells (p=0.003, p=0.002) and SGC7901 cells (p<0.01, p<0.01)8. MGC803 and SGC7901 cells were respectively stimulated by 10%FBS, 10%FBS+IL11(100ng/ml),10%FBS+IL11+S31-201 or 10%FBS+IL11+LY294002.The effect on the migration was assayed by a transwell migration study. The permeable membrane MGC803 cells in 10%FBS+IL11 group was more than that in 10%FBS group (297±15.1 cells/HPF VS 166±11.0 cells/HPF, p<0.05). The permeable membrane MGC803 cells in 10%FBS+IL11 group was more than that in 10%FBS+IL11+S31-201 group and 10%FBS+IL11+LY294002 (297±15.1 cells/HPF VS 165±4.6 cells/HPF,297 ±15.1 cells/HPF VS 178±8.4 cells/HPF,respectively). The permeable membrane SGC7901 cells in 10%FBS+IL11 group was more than that in 10%FBS group (336 ±23.0 cells/HPF VS 166±9.2 cells/HPF, p<0.05). The permeable membrane SGC7901 cells in 10%FBS+IL11+S31-201 group was less than that in 10%FBS+IL11 group (157±18.9 cells/HPF VS 336±23.0 cells/HPF, p<0.05).9. MGC803 cells were stimulated by 10%FBS,10%FBS+IL11, 0%FBS+IL11+S31-201 or 10%FBS+IL11+LY294002 respectively and the effect on invasion of MGC803 cell was evaluated using a matrigel invasion assay. The permeable membrane cells in 10%FBS+IL11 group was more than that in 10%FBS group (110+15.9 cells/HPF VS 41+4.6 cells/HPF, p<0.05). The permeable membrane cells in 10%FBS+IL11+S31-201 group and 10%FBS+IL11+LY294002 group was less than that in 10%FBS+IL11 group (58 ±8.1 cells/HPF VS 110±15.9 cells/HPF,60±11.1 cells/HPF VS 110±15.9 cells/HPF, p<0.05).10 In MGC803 and SGC7901 cells, STAT3 became phosphorylated after 5-min stimulation with 100 ng/ml rhIL-11, and this activation continued for at least 1 h. The inhibitor S31-201 was added to cell culture 30min before rhIL-11 and reduced phosphorylation of STAT3 in MGC803 and SGC7901 cells at the time 5min. In MGC803 cell, ERK1/2 was already phosphorylated in unstimulated cells, and rhIL-11 did not influence their phosphorylation states. In SGC7901 cell, ERK1/2 was only slightly phosphorylated in unstimulated cells, and IL-11 did not influence their phosphorylation state. In MGC803 cell, the AKT1/2 protein was phosphorylated after stimulation for 30 min with rhIL-11, and this activation continued for at least 1 h. The inhibitor S31-201 was added to MGC803 cell culture 30min before rhIL-11 and reduced phosphorylation of AKT. The AKT was not phosphorylated in unstimulated cells, and rhIL-11 did not influence its phosphorylation state.11. After treatment of MGC803 cells with rhIL-11(100 ng/ml) at four different time points (12,24,36 and 72 hours), the changed expression of ICAM-1, MMP2 and MMP9 was not detected by western blotting. After treatment of MGC803 cells with rhIL-11 (100 ng/ml) at three different time points (1,6, and 24 hours), the mRNA expression of COX2, VEGF, HIF-la evaluated by Q-RT-PCR did not change (p=0.975,p=0.108,p=0.886, respectively)12. The MGC803 cells with prolonged stimulation by rhILll(five days) grew loosely and expanded uniformly. Some cells were elongated and no cell mass clusters were detected. The MGC803 cells with stimulation by 0%FBS were detected in thick massive growth pattern and were arranged compactly. After treatment of MGC803 cells with rhIL-11 (100 ng/ml) at 36 hours, the expression of E-cadherin was down-regulated and the expression of vimentin was up-regulated in western blotting and immunecytofluorescent experiments.13. The MGC803 cells were respectively transfected with AKT-siRNA, AKT-siRNA sc, STAT3-siRNA and STAT3-siRNA sc and then were treated by rhIL-11 (100 ng/ml) at 36 hours. The expression of E-cadherin and vimentin was evaluated by western blotting. We found that the expression of E-cadherin was not affected in IL11+STAT3-siRNA group and IL11+AKT-siRNA group. The expression of vimentin was not affected in IL11+AKT-siRNA group. The expression of vimentin in IL11+STAT3-siRNA group was lower than that in IL11+STAT3-siRNA sc group.14. No differences in the genotype distribution of rs1126757 SNP were observed between GC group (GG,8.3%; AG,37%; AA,54.6%)and control group(GG,8.7%; AG,33%; AA,58.3%, p=0.734). No differences in the genotype distribution of rs4252546 SNP were observed between GC group (AA,9.3%;AG,37.0%;GG,53.7%) and control group(AA,7.1%;AG,33.2%;GG, 59.7%, p=0.500). No differences in the genotype distribution of rs1061758 SNP were observed between GC group (AA,22.2%;AG,53.7%;GG,24.1%) and control group(AA,24.9%;AG,49.6%;GG,25.5%, p=0.742). No differences in the genotype distribution of rs2812357 SNP were observed between GC group (CC,19.4%; CT,50.9%;TT,29.6%) and control group (CC,16.7%; CT,60.3%;TT, 23.0%, p=0.212). Compared with the wild-type genotype, rs1126757 (AG+GG) genotypes was not found to be associated with risk of GC in total population (OR=0.985,95%CI:0.454-2.139). Compared with the wild-type genotype, rs4252546 (AG+GG) genotypes was not found to be associated with risk of GC in total population (OR=1.330,95%CI:0.620-2.854). Compared with the wild-type genotype, rs 1061758 (AG+GG) genotypes was not found to be associated with risk of GC in total population (OR=0.860,95%CI:0.516-1.435). Compared with the wild-type genotype, rs1061758 (CT+TT) genotypes was not found to be associated with risk of GC in total population (OR=1.232,95%CI:0.757-2.005). No overall effect of these four SNPs on GC risk was observed in total population adjusted for age, sex, smoking and drinking. No overall effect of these four SNPs on GC risk was observed in subpopulations according to age, smoking and drinking. Compared with the wild-type genotype, rs 1061758 (AG+GG) genotypes was found to be associated with reduced risk of GC in subgroups of female (OR=0.102,95%CI: 0.012-0.895).Conclusion1. IL11RA may play a role in gastric tumorigenesis.2. H. pylori infection may up-regulate the expression of IL11 in chronic gastritis and gastric precancerous lesions and thus promote gastric tumorigenesis.3. The IL11 expression in GC tissues could be used as prognostic indicators.4. IL11 could induce EMT and promote invasion and metastasis of gastric cancer cells through STAT3 and PI3K-AKT pathways.5. The rs 1126757 and rs4252546 in the IL11 gene were not associated with risk of GC in total population. The rs1061758 and rs2812357 in the IL11RA gene were not associated with risk of GC in total population. But the allele change (A'G) in rs 1061758 was associated with a reduced GC risk in the subgroups of females. |
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