| Objective:To detect the levels of human leukocyte antigen-G (HLA-G) in gastric cancer (GC) tissue and cell lines, and analyze the relationships with clinicopathologic features and the roles in tumor immune escape and prognosis. To investigate the potential role of plasma soluble HLA-G (sHLA-G) in the diagnosis and prognosis of GC.Methods:1. One hundred and seventy-nine surgical specimens and corresponding adjacent normal gastric mucous tissues were assessed immunohistochemically for HLA-G expression and tumor-infiltrating regulatory T cells (Tregs). Their correlation with clinicopathologic features and possible prognostic significances were analyzed.2. Thirty surgical specimens and corresponding adjacent normal gastric mucous tissues were collected and the expression of membrane-bound HLA-G were confirmed by western blot to illustrate the difference of HLA-G between gastric cancer and corresponding adjacent normal gastric mucous tissues.3. The levels of HLA-G in human gastric cancer cell lines AGS, BGC-823, SGC-7901, MKN-45, MKN-28 and HGC-27 were detected by RT-PCR, Real-time PCR and Flow Cytometry.4. For transfection treatment, SGC-7901 cells were transfected with HLA-G eukaryotic expression vector (pVITR02-mcs vector-HLA-G) as SGC-7901-G or pVITRO2-mcs control vector as SGC-7901-V using Lipofectamine 2000 according to the manual instructions. Transfectants were screened with hygromycin B. HLA-G expression increased after HLA-G transfection which further confirmed by quantitative Real-Time PCR and western blot. The transfected SGC-7901 cells were cocultured with PBMC directly and indirectly for 24 hours, and CD4+CD25+Foxp3+ Tregs were analyzed by flow cytometry. Moreover, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-y and TNF-a of cocultured supernatants were analyzed by ELISA. The possible molecular mechanisms by which HLA-G contributes to tumor immune escape were evaluated.5. sHLA-G was detected by ELISA in 82 cases with gastric cancer,39 cases with gastric intraepithelial neoplassia,36 cases with atrophic gastritis,52 cases with superficial gastritis and 67 cases of health controls, while the levels of carcino-embryonic antigen (CEA) was also detected simultaneously. The diagnostic value of sHLA-G and its correlations with clinicopathologic features were analyzed.Result:1. HLA-G expression was detected in 49.7% (89/179) tumor tissues and was not detected in corresponding adjacent normal gastric tissues (0/179). In addition, the HLA-G positive expression was significantly correlated with tumor invasion depth, invaded adjacent organs, and clinical stages (P<0.05). However, HLA-G positive expression didn't correlate with age, gender, histological differentiation and Lymph node metastasis(P>0.05, respectively). Kaplan-Meier analysis showed that patients with HLA-G expression had a significantly poorer overall, cancer-specific and disease-free survival than those without HLA-G expression at 5 years after operation(P<0.05, respectively). Moreover, Cox regression multivariate analysis showed that HLA-G expression still retained its significance as independently prognostic factors for unfavorable overall, cancer-specific and disease-free survivals. We also found that the number of tumor-infiltrating Tregs was significantly correlated with invasion depth, lymphatic metastasis, invaded adjacent organs, and clinical stages(P<0.05, respectively). Meanwhile, patients with high Tregs had significantly poorer overall, disease-specific and disease-free survivals than the remaining cases at 5 years after operation (P<0.05, respectively), which demonstrated Tregs contributed to poor prognosis in GC patients.2. To confirm the existence of HLA-G in human gastric cancer, we also detected the HLA-G protein by Western blot. Evidence for HLA-G protein, indicated by the occurrence of a discrete band with a molecular weight of about 39kDa, was detected in gastric cancer and in none of corresponding adjacent normal gastric tissues.3. HLA-G expression was examined in a panel of GC cell lines (AGS, BGC-823, SGC-7901, MKN-28, MKN-45 and HGC-27) by quantitative RT-PCR. Interestingly, HLA-G expression was weak in all these cell lines, which was different from that observed in tumor tissues. These findings were confirmed by flow cytometric analysis. In addition, HLA-G expression increased after HLA-G transfection which further confirmed by quantitative Real-Time PCR and western blot.4. There was a significant increase in the percentage of CD4+CD25+Foxp3+ Tregs after PBMC direct coculture with SGC-7901-G rather than with SGC-7901-V. In addition, notably, levels of IL-6 and IL-10 were significantly higher in supernatants of PBMC direct coculture with SGC-7901-G. No significant differences were found between two groups in terms of IL-2, IL-4, IL-8 and IFN-γlevels. TNF-αlevel of SGC-7901-G group was lower than SGC-7901-V group.5. The level of sHLA-G was significantly higher in gastric cancer group than that in gastric intraepithelial neoplassia group, atrophic gastritis group, superficial gastritis group and health control group (P<0.001, respectively). The level of sHLA-G was significantly higher in gastric intraepithelial neoplassia group than that in health control group(P<0.05). The levels of CEA in gastric cancer group was significantly higher than that in health control group, superficial gastritis group, atrophic gastritis group, and gastric intraepithelial neoplassia group (P<0.01, respectively). The level of sHLA-G in gastric cancer patients was significantly correlated with age and tumor size(P<0.05, respectively), while there were no significant correlations between the level of sHLA-G and other clinicopathologic factors were detected (P>0.05, respectively). ROC curve analysis showed that AUC for sHLA-G was 0.814, which was significantly higher when compared with the AUC of CEA(0.692, P=0.01). The AUC of sHLA-G and CEA combined was 0.846, which was significantly higher when compared with the AUC of sHLA-G or CEA individually (P<0.001). The results indicated that sHLA-G, as a diagnosis indicator in early-stage, had better values than CEA. Plasma sHLA-G level could be a highly specific and sensitive index to diagnose gastric cancer.Conclusion:1. Expression of HLA-G in gastric cancer is significantly higher than that in corresponding adjacent normal gastric tissues. In addition, HLA-G positive expression had a significantly poorer overall, cancer-specific and disease-free survival than those without HLA-G expression at 5 years after operation and still retained its significance as independently prognostic factors for survivals. All the results indicated that HLA-G may play an important role in the development of gastric cancer.2. The number of infiltrating Tregs in gastric cancer was significantly higher than that in corresponding adjacent normal gastric tissues. The sections with negative expression of HLA-G barely showed tumor-infiltrating Tregs, while sections with positive HLA-G expression showed lots of tumor-infiltrating Tregs. The results indicated a significant positive correlation. In addition, the number of tumor-infiltrating Tregs was significantly correlated with invasion depth, lymphatic metastasis, invaded adjacent organs, and clinical stages. Meanwhile, patients with high Tregs had significantly poorer overall, disease-specific and disease-free survivals than the remaining cases at 5 years after operation, respectively, which demonstrated Tregs contributed to poor prognosis in GC patients.3. The coculture experiment showed overexpression of HLA-G in GC cell line significantly enhanced the frequency of Tregs when GC cells were directly cocultured with human peripheral blood mononuclear cells. Meanwhile, the balance of Thl and Th2 cytokines was also substantially modulated in the coculture system. The results indicated that HLA-G expression was involved in tumor evasion by increasing the frequency of infiltrating Tregs locally and modulated the balance of Thl and Th2 cytokines. Thus, HLA-G might be a promising predictor for disease prognosis and a possible novel target for immunotherapy in GC patients.4. Compared with CEA, sHLA-G had ascendancy in the diagnosis of early-stage in gastric cancer. sHLA-G might be a potential biomarker for the early diagnosis of gastric cancer patients clinically. |
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