The Expression Of FoxP3and Its Role In Gastric Cancer

Background and Objective:Forkhead box protein3(FoxP3) was used to be defined as a key transcriptional factor of regulatory T cell (Treg), contributing to the occurring and development of Treg, which associated with clinicopathological characteristics and prognosis. Recently, FoxP3has been identified to be expressed in many kinds of tumors and been in favour of clinical assessment. Previous study demonstrated that tumor cell could transfer CD4+T into Treg, resulting in immune escape. However, it is still unclear about the act way and mechanism during the interaction between tumor cell and lymphocyte, both of which expressed FoxP3. To present, FoxP3gene’s function and its contribution to the biological behavior of gastric cancer (GC) cell remains poorly understood. Only in breast cancer and prostatic carcinoma, it was noted as an anti-oncogene which depressed the expressions of several oncogenes and inhibited tumor growth. Based on these previous researches, the aim of the current study is to detect and analyze the expression of FoxP3in the process of tumor development, and to study the mechanism of the interaction between tumor cell and lymphocytes, as well as to explore FoxP3gene’s function and related signal passway, so as to explain the mechanism of FoxP3in the carcinogenesis and progression in GC.Methods:1. Quantities real-time polymerase chain reaction (qRT-PCR) and Immunohistochemistry (IHC) techniques were used to detect the mRNA and protein statuses of FoxP3in the tissues of gastric precancerosis and cancer. Immunofluorescence technique was performed to evaluate the expression and location of FoxP3in several GC cell lines. IHC was applied to determine the expression of FoxP3in GC cells and lymphocytes in the GC tissue microarray, respectively. Moreover, in the GC tissues and tissue microarray, the associations between the positive expression of FoxP3in either cancer cells or lymphocytes and tumoural clinicopathological characteristics were analyzed by chi-square test and their significance of prognostic assessment was analyzed by kapar-meier and Cox regression analysis.2. The direct coculture and indirect coculture systems were established to study the interacted manners. Cell-IQ technique and MTT assay were performed to monitor and detect the growth and proliferation of GC cells and lymphocytes. Quantities RT-PCR and Western blot were applied to determine the changes of the transcripts and expression of FoxP3and related molecules after cocultures. Enzyme linked immune sorbent assay (ELISA) technique was performed to measure the secretion of TGF-β1and TGF-β2cytokines in cell supernatant after cultures.3. In vitro, the plasmid of FoxP3-shRNA was transfected into GC cells, after drug-selecting and monoclone-picked, the stable expression FoxP3cells were indentified. CCK-8assay was used to detect the growth and proliferation of GC cells. Terminal Transferase dUTP Nick End Labeling (TUNEL) assay and flow cytometry were performed to determine cell apoptosis and cell cycle of the transfected cells. The soft agar colony formation assay was performed to evaluate the ability of colony formation. MTT assay was used to measure the sensitivity to chemotherapy drugs. In vivo, the genetic function was further confirmed by subcutaneous tumor formation experiment in nude mouse.4. Quantities RT-PCR technique was used to detect the transcripts of TGF-β and related molecules. after the GC cells transfected with FoxP3gene, ELISA technique was used to test the secretion of several cytokines. Western blot technique was applied to determine the changes of some apoptosis-associated proteins and the molecules of NF-κB related signal passway.Results 1. In GC tissues, tumoural FoxP3strongly expressed in cytoplasm and weakly/none expressed in nucleus, while lymphocytes expressed in nucleus. In GC cell lines, Foxp3located both in cytoplasm and in nucleus. FoxP3mRNA levels were increased in the sequence from precancerosis, early GC to advanced GC (P=0.001). FoxP3mRNA levels were significantly higher in tumoural than peritumoural tissues (P=0.003). Its levels positively related with TGF-β1(P=0.020, r=0.351) and HER2(P=0.002, r=0.443) respectively. Moreover, corresponding FoxP3protein expression was increased in the same sequence, positively correlated with the tumor progression (P=0.008). However, the tumoural FoxP3expression and the density of Treg have no significant association with age, sex, TNM stage and lymph node metastasis in tumoural tissues. In the tissue microarray composed of135cases of GC, prognostic analysis showed that the patients with positively tumoural FoxP3+had a longer survival time and the prognosis was good(P=0.042), while the patients with high count of Treg showed a shorter survival time and the prognosis was poor (P=0.034). It was further showed that tumoural FoxP3+was a benefit prognostic factor and Treg was a hazard prognostic factor by Cox regression analysis.2. The interaction between GC cells with lymphocytes inhibited the growth of cancer cells; this effect was more remarkable in the direct coculture compared to the indirect one. The mRNA and protein levels of FoxP3in GC cells after cocultures. TGF-β1and TGF-β2cytokines in cell supernatant were increased in the direct coculture compared to the indirect one.3. After transfected with FoxP3gene, compared to the vector-transfected control, the GC cell growth was inhibited, cell apoptosis was induced and the ability of invasion was decreased and the sensitivity to anti-tumor drugs was increased.In addition, up-regulation of FoxP3can inhibit the growth of GC in vivo. 4. Up-regulation of FoxP3induced pro-apoptosis proteins and reduced anti-apoptosis proteins. Meanwhile, it inhibited the phosphorylation of canonical NF-κB signaling and non-canonical NF-κB signaling.Conclusions:1. FoxP3is overexpressed in GC tissues. It locates both in tumor cells and lymphocytes. The expression of FoxP3in GC cells and the high density of Treg have different clinical significance in prognostic assessment.2. Although the expression of FoxP3was increased in GC tissues, it correlated with good prognosis. It may be associated with the FoxP3gene’s function of inhibiting growth and inducing apoptosis, and with several related molecules, such as TGF-β and HER2, as well as with the interaction to lymphocytes in tumor microenvironment. The increased sensitivity to chemotherapy drugs also contributes to it.3. The iteration between GC cells and PBMCs can inhibit tumor growth, dependent on the direct contact way more than on the cytokine-dependent way.4. Up-regulation of FoxP3gene can inhibit cell growth and induce apoptosis in vitro and in vivo, mediated by NF-κB cell signal passway.