The Function And Mechanism Of TGF-β In Tumor Escape Mechanisms Of Gastric Cancer

Background:The early symptoms of gastric cancer patients without a certain specificity.Nearly half of the patients in the treatment of patients with advanced to the disease.Although the relevant treatment progress faster, but the prognosis is still poor.At present, scientists have studied the role of cytokines, especially growth factor transforming (TGF), which is an important part of the family. Transforming growth factor beta (TGF-β) is a new cytokine, which has a broad range of biological activities. Almost all of the cells are controlled by a series of life events, including proliferation, differentiation, apoptosis, signal recognition, and embryonic development. The present study suggests that, transforming growth factor (TGF-β) produced by T cells has been shown to be an important factor in the study of antitumor immune responses. The pathogenesis of gastric cancer is influenced by many factors, which is related to the development of immune system’3]. In addition to the inherent effects of common cancer cells and stromal cells, it also includes tumor microenvironment that enables the formation of immune cells in gastric cancer。In the past ten years, the diagnosis and treatment of gastric cancer, patients have a relatively long survival time, but the high metastasis rate of gastric cancer has affected the further treatment of gastric cancer. So far, the clinical and pathological characteristics of gastric cancer, tumor histology, lymph node metastasis research is limited, and there is considerable controversy. Commonly used methods of diagnosis of gastric cancer, although there are pathological diagnosis, physical diagnosis (imaging diagnosis), Tumor associated markers of the serological examination and other advanced diagnostic methods, but these methods or operations are complex, expensive, or need to perform the operation, causing damage to the body。The most important is often diagnosed as gastric cancer symptoms。And the pathogenesis of gastric cancer is a multi factor, which is formed by the interaction of multiple factors, including genetic, tumor suppressor genes, oncogenes, growth factors, and gastric cancer related cell adhesion molecules. Therefore, the molecular mechanism of gastric cancer has important clinical value in the treatment of gastric cancer. To study the molecular mechanism of gastric cancer, and to explore the mechanism of invasion and metastasis of gastric cancer, and to explore the molecular mechanism of invasion and metastasis of gastric cancer, and to improve the survival rate of patients with gastric cancer。Previous studies have indicated that TGF-β cells were found to inhibit the anti tumor immune response by T cells in the gastric cancer research , which shows that TGF-βis an important factor in the immune surveillance of gastric cancer. Although the immune escape mechanism of TGF-(3is not clear, some studies have shown that TGF-P is associated with human leukocyte antigen (HLA-G)。HLA-G levels in gastric cancer patients were associated with low survival rates. This suggests that HLA-G may be involved in immune escape, and studies have demonstrated that cells are associated with increased Foxp3+ regulatory T cells by inhibiting CD8+T lymphocyte mediated cells.HLA-G and miR-152 were analyzed by computer analysis of HLA-G3’-UTR,, and microRNA. We found that the level of HLA-G in gastric cancer patients was positively correlated with TGF-β. In addition, miR-152 further confirmed the role of TGF-β in the regulation of immune escape. MiR-152 indicated that may inhibit the over expression of HLA-G. MiR-152 leads to increased NK cell mediated lysis, which suggests that miR-152 may act as an immune system enhancer for the function However, it is not yet known how miR-152 expression changes in gastric cancer cell HLA-G, and the changes of and miR-152 in the immune escape of TGF-β induced by HLA-G.. Based on the above problems, we have not yet resolved, so we have to investigate whether the expression of HLA-G in gastric cancer cells, miR-152 and TGF-β, miR-152, HLA-G, and TGF-β, and to study the immune escape mechanism. This study is to explore the new molecular mechanism between tumor immunology and cell molecular biology, which has important scientific significance. Have the following two parts of the discussion.The expression levels of HLA-G TGF-β in gastric cancer tissues of 20 patients with gastric cancer were detected by Immuno Sorbent Assays-G (Enzyme-Linked). It was found that the expression level of TGF-β in gastric cancer tissues was significantly higher, and the level of was highly correlated with the level of HLA-G in tumor tissues and peripheral blood. Then, we studied the function of and TGF-βand the results showed that TGF-βould up regulate the mRNA level and protein level of HLA-G in vitro. On the other hand, bioinformatics analysis showed that the interaction between miR-152 and TGF-β, suggesting that miR-152 may be involved in the regulation of HLA-G expression. Our histological studies showed that the expression of miR-152 in gastric cancer tissues was changed, and the expression of miR-152 was negatively correlated with the expression of TGF-β. Our in vitro experiments confirmed miR-152 on gastric cancer cells of HLA-G expression there is a negative regulatory role, the mutation experimental study showed that TGF-βoverexpression can eliminate the miR-152 induced by HLA-G 3-UTR activity decreased。Therefore, we believe that TGF-Pmay be involved in the immune escape of gastric cancer by inhibiting miR-152 expression in gastric cancer HLA-G. Not only that, this study also shows that the TGF-βis used to improve the prognosis of patients with gastric cancer, improve the survival rate of potential.The first part Experimental study of HLA-G expression in gastric cancer cell by TGF-p inducedObjective:Though induce TGF-P in gastric cancer cell line to study the expression of HLA-G in gastric cancer tissues.Research method20 cases of gastric cancer tissues and adjacent tissues were collected from Oilu Hospital of Shandong University, and the peripheral blood samples were collected from 1 days before operation. The study was approved by the hospital ethics committee, and all patients were included in the operation before the signing of informed consent.1 Research data sources:Clinical data from July 2013 to September 2012 in the treatment of gastric cancer patients with gastrointestinal surgery in Hospital. Patients (15 men,5 women) who were selected to meet the requirements of patients with gastric cancer underwent surgical operation. Experimental group see text. Diagnosis and staging according to the international classification standards (TNM).2 The tissue specimens and special apparatusIn gastric cancer patients after surgery collected standard in tumor tissues and corresponding normal mucosa tissue cryopreservation and after using conventional paraffin embedded and fixed in the formalin solution taken tumor sections. To be used for immunohistochemical staining and PCR technique. Peripheral blood samples were collected from 1 days before operation, and the EDTA plasma was used for the future use of-80. EDTA plasma or cell culture supernatant-after 48 h of incubation in the absence of serum culture medium preparation reagent. TGF-βlevels were determined by using a TGF-βKit (Systems HLA-G,125 MN, R&D), respectively, in accordance with the manufacturer’s description of ELISA Minneapolis (R&DSystems,125, Minneapolis, MN)) and sHLA-G (Exbio) kits (Biovendor and, Prague, CZ), respectively.,3 Preparation of tissue pathologyThe peripheral blood samples with EDTA plasma treatment after the village into the -80℃ liquid nitrogen freezing standby. Detection of EDTA plasma samples by ELISA method. SHLA-G kit was used to detect the concentration of soluble HLA-G.4 The peripheral blood samples with EDTA plasma treatment after put in liquid nitrogen freezing standby-80℃ The concentration of soluble HLA-G in peripheral blood was detected by using sHLA-G kit.5.ELISA detect the concentration of HLA-G and TGF-β in gastric cancer cells.Pre operation preparation:according to the pre experiment selected the appropriate enzyme labeled antibody dilution. Dilution specific antibodies or immune globulin fraction with PBSsolution, the configuration of the capture antibody solution (the final concentration of 0.2-10 g/ml). In accordance with the requirements of manufacturers to use Kit ELI5A as well as sHLA-G kit for determination. Use colorimetric method calculation results judgment (enzyme labeling instrument was used to measure the OD). Absorption determination: application of TGF-β ELISA kit and sHLA-G kit according to the different concentrations used to detect changes in the amount of TGF-βlevels and HLA-G concentration. Data processing, mapping, and the linear equation6. TGF-β kit and sHLA-Gkit were used to detect the concentration of HLA-G and TGF-β in gastric cancer cells.SGC7901 And BGC823 cells were prepared，TGF-β,and the expression levels of HLA-G were detected by, sHLA-G kit and TGF-β kit.HLA-G and TGF-β in gastric cancer cells at different concentrations of (2.5,5, or 10ng/ml TGF-β). Results are shown in figure 2.7 Statistical analysisFor TGF-Pand HLA-G concentrations, the relevant data correlations (Pearson correlation analysis) was used to evaluate the data of different concentrations of TGF-β. Using the Mann-Whitney U test, one-way ANOVA or chi square difference test method to analyze 2 groups. Statistical analysis between SPSS 17. P<0.05 thought there was statistically significant.Research results1 Expression of TGF-Pand HLA-G in the tissues of gastric cancer. To investigate the relationship between TGF-βand HLA-G levels in patients with gastric cancer, and to analyze their concentrations, from peripheral blood samples from patients with gastric cancer. A strong positive correlation was analyzed by Pearson’s regression. R2=0.5455; P< 0.001.2 ELISA experimental analysis showed that the HLA-G level was higher than that of at 48 hours after treatment with the same method。*P<0.05** P<0.01。3 Experimental results show that the HLA-G expression gradually increased with the increase of TGF-βconcentration and treatment time.Conclusion1 The expression of HLA-G and TGF-β in gastric cancer tissues was highly expressed, and the expression of the two groups was statistically analyzed.2.TGF-βexpression of HLA-G was up-regulated in gastric cancer. The expression of TGF-βand HLA-G in gastric cancer tissues were significantly positive correlated with the expression of TGF-p.3 The expression of HLA-G and TGF-βin gastric carcinoma is not only related to the development and metastasis of gastric cancer, but also can be used as an indicator of prognosis of gastric cancer patients.The second part To study the effect of TGF-β on the immune escape of gastric cancer by inhibiting miR-152 and its mechanism Research purposesResearch purposes:To investigate the relationship between HLA-G miR-152 and TGF-β in gastric cancer cells, and to explore the possible mechanism of TGF-β in the immune escape in gastric cancer cellsResearch method:In the first experiment, the cells were treated by cell recovery and cell digestion. The cells were cultured and the next step was achieved.1 Conventional cell recovery and cell culture, passageFrom the liquid nitrogen tank removed cell lines cryopreserved in vitro, until the cell recovery into 37 "Cwater bath and two human gastric cancer cell SGC7901 and MGC-823 with containing 10% fetal bovine serum (fetalbovineserum FBS), the antibiotic (1% streptomycin and penicillin) of RMPI-1640 culture medium and cultured in 37 ℃, containing 5% CO2 saturated humidity constant temperature incubator. Then remove the cell. Centrifugation, discard the upper liquid, add the right amount of cell culture solution, then inoculated in the culture dish. The cell density was higher when the cell density was higher. The cells needed for the experiment were third generations after the recovery. Cell supernatants were collected after stored for standby.2 Cell transfection, plasmid transfectionAfter the cell recovery will SGC7901 and BGC823 cell line with 1 × 105/ml density to 6-well plate were given different concentrations of TGF βstimulation, and in cells was detected 24h or 48h after the expression of HLA-G mRNA and cell supernatant HLA-G expression. The transfection was carried out with the cationic liposome method (according to the manufacturer’s instructions). All plasmid vectors for transfection were extracted by Midiprep kit DNA after amplification. SiRNAs and siRNA (si-NC) purchased in Invitrogen company. After 24 hours of incubation, mimics miR-152, miR-152 inhibitor expression plasmid and interference plasmid were transfected into gastric cancer cells in the Opti-MEM medium. Specific transfection method with reference to Lipofectamin 2000 transfection kit.3 Luciferase assayWith the PGL3 vector containing HLA-G 3 UTR luciferase reporter plasmid. Primer see text. PGL3-HLA-G and PRL-TK were transfected with mimics miR-152, inhibitor miR-152 and negative control RNA relative.24 hours after transfection, using dual luciferase 1000 detecting luciferase activity analysis system. All experiments were performed at least three times to demonstrate the results.4.RNA was isolated, qRT-PCR to detect mRNA TGF-β and HLA-GGastric cancer cell total RNA was extracted by Trizol method, in order to detect the expression of HLA-G mRNA and TGF-β, using superscript III reverse transcriptase according to the product specification will oligo DT primers calibration RNA reverse input cDNA and used phosphoglycerate dehydrogenase (GAPDH) was used as a reference. In order to detect mature miR-152, RNA mirVana miRNA Isolation to extract kit, with minor RNA U6 as reference. Real time quantitative polymerase chain reaction in ABI 7300 SYBR ExTaq enzyme premix. Real time fluorescence quantitative PCR (qRT-PCR) detection and data collection were performed by using ABI 7300 real-time fluorescent quantitative kit. Takara. And the relative quantitative analysis of the experimental data is carried out by comparing the threshold method.5 TGF-βand HLA-G concentrations were detected and analyzed by miR-152.Using ELISA method to detect the culture supernatant of kit ELISA TGF-|3and sHLA-G TGF-βwere used to detect the concentration of soluble HLA-G. Then detect the concentration of miR-152 and analyze the relationship between them.6 Statistical analysisDifferences between groups were compared using the one-way AVOVA (single factor variance analysis and Mann Whitney U test or chi 2 test method was used to analyze the between group differences. Using SPSS 17.0 statistical analysis were analyzed using the Mann Whitney U test or chi square test, statistical analysis using SPSS 17 computer software, P< 0.05 is considered to have significance statistical.Research results1 The 24h (2.5,5 or 10ng/ml) was found to increase the expression of mRNA and BGC823 in the same time, and the HLA-G and SGC7901 were also detected by using different concentrations of TGF-β. Figure 2B2 The different concentrations of miR-152 were detected in TGF-pcells and cell lines. In vitro TGF-βinduced by miR-152 was significantly reduced) MiR-152 in gastric cancer cells. (P<0.01) Figure 3A。3 Changes in BGC823 and SGC7901 cells in gastric cancer and the-expression of HLA-G. A similar trend (Figure 3) was observed in the soluble HLA-G concentration after transfection of 48 h.4 Comparative analysis of miR-152 and TGF-in the regulation of HLA-G expression. In Figure 7, the relative expression of HLA-G (Figure 4A), concentration (Figure 4B) and HLA-G-3 UTR activity (Figure 7C) found. The level of HLA-G was down regulated by TGF-β, and was up-regulated by miR-152. After 24 h of Mut-HLA-G, the 3’UTR non translation region was changed by HLA-G (7C), and no obvious rule was observed at the same time (Fig.7E, F).5 MiR-152 mimics, inhibitor negative, control RNA and mimics control were used to detect the miR-152 miR-152 and miR-152 can be used to regulate the expression of HLA-G. Inhibition of HLA-G expression by TGF-β.**P<0.01,*P<0.05.6 After detection, HLA-G expression and TGF-βexpression were up-regulated, while the addition of mimics miR-152 was partly down regulated. Figure 4A B,. *P<0.05,**P<0.01.Figure was used to find the relationship between the concentration of HLA-G and miR-152.R2=0.5267 P;< 0.001. At the same time, the relationship between TGF-pconcentration and miR-152 wasProportional relationship; R2=0.5944P< 0.001.Conclusion1 Studies have found that different concentrations of TGF-βcan produce different concentrations of miR-152 and vice versa, which shows that there is interaction between the two.2 Through the analysis above, it can be found that miR-152 can promote the expression of TGF-P in gastric cancer cells. The expression level of miR-152 in gastric cancer tissues was in inverse proportion to TGF-β(P< 0.001).3 There was a significant negative correlation between.HLA-G expression and miR-152 concentration (R2=0.5267; P< 0.001). In this experiment, miR-152 was found to have low expression in gastric cancer cell line, and the experiment showed significant difference (P<0.001).4 Results show that TGF-P can induce the expression of HLA-G in gastric cancer cells after inhibition of miR-152 transcription activity. This plays an important role in the activation of gastric cancer cells and has important significance for further study on the immune escape mechanism of TGF-pin gastric cancer.Significance:It was found that the expression level of HLA-G and TGF-pin gastric cancer could be increased by inhibiting miR-152 transcription activity, which may reveal the immune escape mechanism of gastric cancer TGF-. This study is also the first time to reveal the relationship between miRNA levels and TGF-p,HLA-G, for the forward looking for new target for gastric cancer, new biological technology has important theoretical and clinical significance.