Effects Of TAM On GPER-mediated Proliferation、Migration And Apoptosis In Cancer Associated Fibroblasts

Objective To construct lentivirus vector targeting GPER gene and explore the effects of tamoxifen (TAM) on the cell proliferation,migration and apoptosis of cancer-associated fibroblast (CAF) mediated by G-protein coupled estrogen receptor (GPER).Methods 1.Effective target sequences that target at GPER and negative control were designed,then synthetized the oligonucleotide sequences.After annealing of the complementary strands, the DNA fragments were connected to the GV115 vectors by double digestion with Age I and EcoR I to construct the lentiviral vectors which expressed short hairpinRNA. The lentiviral vectors were identified by PCR and DNA sequencing. Recombinant lentivirus and control were extracted after transfecting HEK293T with the recombinant vector and helper vectors. After infection of CAF cells with the GPER lentiviral vector under the best interfering condition, GPER expression was confirmed by western blot.2.CAFs were divided into four groups including negative control (NC) group,GPER-RNAi group, negative control combined with tamoxifen group and GPER-RNAi combined with tamoxifen group. CCK-8 assay was used to detect the proliferation. Cell cycle was measured by flow cytometry. Transwell assay was used to evaluate the migration.Annexin V-fluorescein isothiocyanate/propidium iodide (annexin V-FITC/PI) combined with flow cytometry was used to detect the apoptosis.Results 1.The recombinant lentiviral vector harboring siRNA targeting the GPER gene was successfully transfected into HEK293T cells. Western blotting results showed that the protein expression of GPER was significantly reduced in CAF(P<0.05).2.The Proliferative Index(PI) of each group were (30.76±7.4)%, (32.54±3.5)%, (43.61±4.4)%(P=0), (34.93±6.8)%. The results of CCK8 assay showed TAM promoted cell growth of CAF and increased the cell number to (172.16±10.8)%(p=0) of NC group after 72h cultivation. There were no significant difference between The cell number of GPER-RNAi group(p=0.10),GPER-RNAi combined with tamoxifen group (p=0.43) and NC group. The results of apoptosis ratio in each group: (29.10±1.4)%,(28.27±1.3)%,(9.62±0.91)%,(31.09±1.2)%. The apoptosis ratio in GPER-RNAi group was no change than negative control group. The apoptosis ratio in negative control combined with tamoxifen group was obviously lower than that in negative control group and GPER-RNAi combined with tamoxifen group (P<0.05) by Flow cytometry combined double staining. The TAM-treated CAF cells were spread more under microscopy at 12edhour after the migrated more in transwell assay(99.50±4.80vs 40.75±3.59,P=0.002). But the migration of GPER-RNAi group was no obvious difference with negative control group and GPER-RNAi combined with tamoxifen group.Conclusion GPER-siRNA lentivirus vector has been constructed successfully, and it can effectively silence the GPER expression in CAF cells. TAM triggers GPER to accelerate cell proliferation, migration and decreased cell apoptosis of CAF.