GPER Protein Mediated IL-6/STAT3 Signaling Pathway Activation In Endometrial Carcinoma

Objective: Endometrial carcinoma is an epithelial malignant tumor in uterus,one of the most common malignant tumor in female reproductive system. According to the clinical and pathological observation,endometrial carcinoma is divided into 2 types: type?and type?. Type?endometrial carcinoma is estrogen-dependent. The most common pathological type is endometrioid adenocarcinoma, accompanied by early endometrial hyperplasia and(or) endometrial intraepithelial neoplasia, on the stimulation of high level estrogen. Type?endometrial carcinoma is estrogen-independent, and mainly occurs in the atrophy or the resting endometrium. The main pathological types include serous carcinoma, clear cell carcinoma and sarcoma. Type ?endometrial carcinoma is prone to deep myometrial invasion and lymph node metastasis, with large cell atypia, and rapid progress. It's signal transduction pathway is a hot pot in domestic and foreign research. How estrogen induces type?endometrial carcinoma needs further research.G protein-coupled estrogen receptor(GPER) is a membrane-bound estrogen-binding protein, different from classical estrogen receptor. It mediates the effect of estrogen by activating downstream of rapid non-genomic signaling pathway, including MAPK/ERK, PI3K/AKT, c AMP, calcium ions flow et al, then plays malignant biological effects about cell growth, migration, invasion, and angiogenesis. It is widely involved in regulating body physiological activities, by mediating rapid non-genomic effects through estrogen and its related compounds. It arouses wide attention about its structure, location, signal transduction pathways and estrogen-related diseases.Inflammatory microenvironment is one of important characters of tumor. IL-6(interleukin-6) is named as T cell replacement factor by Muraguch, secreted by lymphocytes(such as T cells, B cells), and nonlymphocytes(such as fibroblasts and macrophages). Many antigen and nonantigen substances induce the secretion of IL-6, such as bacterial endotoxin, some Chinese medicine monomer and cytokines, etc. Signal transducers and activators of transcription 3(STAT3) is a class of bifunctional molecule, either cellular signal transduction, and can activate gene transcription. IL-6 activates its downstream STAT3, joining tumor induction and maintenance of inflammatory microenvironment to promote tumor development. Studies have shown high levels of IL-6 in type?and type?endometrial cancer patients, and the level of IL-6 in type? endometrial cancer higher than in type?, which suggested that IL-6 was involved in the regulation of endometrial cancer type ?. Some scholars found that Estrogen can through the MAPK/ERK signaling pathway to promote the expression of IL-6 in the cells.Therefore, it is speculated that the regulation of GPER to IL-6/STAT3 signaling pathway exist in endometrial cancer.The study selected three cell lines as the research object, including Ishikawa cell line whose estrogen receptor is positive?HEC-1A cell line whose estrogen recepter is low expressed and KLE cell line whose estrogen recepter is negative, using estrogen17-?diol(E2), GPER specific agonist(G1), GPER antagonist(G15). And the content of IL- 6 in cell culture supernatant and GPER, P-ERK, P-STAT3 protein expression level were measured. To explore the regulation of GPER/ERK on IL-6/STAT3 signaling pathway, and it's effect on the development of endometrial cancer and the possible mechanism.Methods:1 Cell culture: The Ishikawa cells were cultured in nutrient solution of 1640, and the HEC-1A and KLE cells were cultured in nutrient solution of Mc Coys 5A in vitro, including 10% newborn calf serum, 100U/ml penicillin and 100U/ml streptomycin, at 37?, 5% CO2 saturated humidity incubator. The cells were digested by 0.25% trypsinase and then subcultured. Cells adhered to the bottom in monolayer and were chosen to be used in the test at logarithmic growth phase.2 ELISA was used to detect IL-6 secretion in cells supernatant. To cultivate the bottle after cell growth to 80%~90%, replaced the serum free medium hunger for 24 h, used E2, G1 and G15 separately to deal with three kinds of cells 24 h at the same concentration of 10-6mol/L respectively. Each group adjusted the DMSO to the same. Finally the control group, E2 treatment group, G1 treatment group, E2+G15 treated group, G1+G15 treatment group were gotten. Cell supernatants were collected and stored at-80?refrigerator. Aaccording to ELISA kit operating instructions, the samples was tested. The experiment was repeated at least three times.3 Western Blot was used to detect the influence of 17-?-estradiol(E2)?G1 and G15 on the expression of GPER?P-ERK and P-STAT3 protein on the influence of 17-?-estradiol(E2)?G1 and G15 in Ishikawa cells, HEC-1A cells and KLE cells.Results:1 E2 and G1(GPER special activator) induced the expression of IL-6 in three kinds of cells Ishikawa, HEC-1A and KLE, by activating membrane estrogen receptor GPER.ELISA tested the IL-6 level in three kinds of cells supernatant after E2 and G1 treatment. The results showed that E2 and G1 significantly promoted IL-6 secretion in Ishikawa cells supernatants after drug treatment for 24 h, and the effect of E2 were stronger than G1(P<0.05). E2 and G1 induced IL-6 secretion in HEC-1A and KLE cell supertanant, and the effect of G1 were stronger than E2(P <0.05).The above results showed that the E2 and G1 promoted the expression of IL-6 in three kinds of cells by activating GPER.2 GPER specific antagonist G15 inhibited the secretion of IL-6 in Ishikawa, HEC-1A and KLE cells.ELISA results showed that the level of IL- 6 was significantly inhibited in the E2+G15 group and in the G1+G15 group(P<0.05), especially much obvious in the HEC-1A and KLE cells. It suggested that the induction of IL-6 level in cell supernatant by E2 and G1 worked mainly through GPER.3 The expression of GPER?P-ERK and P-STAT3 protein stimulated by E2 and G1 measured by Western Blot.GPER, P-ERK, P-STAT3 proteins were expressed in the three kinds of cells. The level in KLE cells was the highest and the differences were statistically significant. After the three cells were incubated with E2, G1 at the concentration 10-6mol/L for 0 min, 15 min, 30 min, 45 min, 1h, 2h, Western Blot was done. The results showed that: In the Ishikawa cells, E2 promoted the expression of GPER, P-ERK, P-STAT3, starting at 15 min, reaching the highest levels at 30 min(P<0.05). G1 promoted the expression of GPER, no obvious effect to P-ERK?P-STAT3 level. The above results indicated that E2 worked through ER, nor GPER. In HEC-1A and KLE cells, E2 and G1 promoted the expression of GPER, P-ERK, P-STAT3, reaching the highest levels at 15 min(P<0.05), especially the effect of G1 much obvious. The differences were significant.4 The expression of GREP, P-ERK, and P-STAT3 protein stimulated by E2 and G1 on the influence of G15 in three types of endometrial carcinoma cells detected by Western Blot.Useing E2+ G15?G1+ G15 to deal with three cells separately for different time, Ishikawa cells for 30 min, HEC-1A and KLE cells for 15 min. The results showed: G15 could reduce the protein expression of GPER?P-ERK?P-STAT3 in cells stimulated by E2 and G1 significantly in HEC-1A, KLE cells. Where in Ishikawa cells, G15 could reduce the protein expression of GPER, no obvious effect found about the level of P-ERK?P-STAT3(P>0.05). It indicated that GPER?P-ERK?P-STAT3 were in the same signal pathway in HEC-1A?KLE cells.Conclusions:1 E2 and G1 promote the expression of IL-6 through the activation of membrane estrogen receptor GPER in three cells, especially obvious in HEC-1A and KLE cells, type? endometrial carcinoma cells.2 GPER mediates the estrogen's "The non-transcription effect", and through the GPER regulating inflammation of IL-6/STAT3 signal.3 In type ? endometrial carcinoma, GPER specific antagonist G15 can significantly inhibit GPER mediated IL-6/STAT3 signaling pathway. Therefore GPER and its antagonist G15 may become the new target and new targeted drugs for treatment of type ? endometrial cancer.