GPER-Mediated HMGB1 Exocrine In Cancer-Associated Fibroblasts Promoted Breast Cancer MCF-7 Cells Autophagy And Proliferation

Objective: To explore the effect of activating G protein coupling estrogen receptor(GPER)induced HMGB1 exocrine in cancer-associated fibroblasts(CAF)promoted the autophagy level and proliferation of breast cancer MCF-7 cells.Methods: Through the analysis of the early stage of mRNA of chip data,CAF cells deal with TAM and TAM joint with GPR30 specific inhibitors G15,select the expression differences of production factor,pick out the cytokines HMGB1,and qRT-PCR and Western blot method validation results chip.Constructed the target of shRNA lentivirus to interfere the GPER gene,infected the CAF cells,RT-PCR and Wstern blotting were usded to test the expression of GPER mRNA and protein in CAF-shNC cells、CAF-shGPER cells.The above two group cells treated with specific agonist G1,and respectively to get G1+ CAF-shNC group and G1+CAF-shGPER group,By RT-PCR,Western blotting and ELISA assay to detect the expression levels of HMGB1 mRNA and protein,and the secretion of HMGB1 in the above each group cells and its conditioned medium.Adding different concentrations of exogenous recombinant protein HMGB1 and collect the above each conditioned medium and above each medium respectively to join HMGB1-neutralizing antibody treatment MCF-7 cell,the expression levels of Beclin1,P62,LC3 in MCF-7 cells and cells proliferation were detected by Western blotting and CCK8 assay.Results: Found in the preliminary results of gene chip GPER mediated the CAF cells secrete a variety of cytokines,and cytokines HMGB1 in CAF TAM treatment cells HMGB1 mRNA and protein expression up-regulated(P < 0.01),and for this kind of effect can be G15 block(P < 0.01).stable transfection of CAF cells by shGPER lentivirus,the expression of GPER mRNA and protein were significantly inhibited(P < 0.01).GPER activated by G1 that up-regulated the expression of HMGB1 mRNA and protein,and increased the secretion of HMGB1 cytokines in CAF-shNC cells(P < 0.05).However,these above effects were significantly suppressed in the G1+CAF-shGPER group.Interestingly.Exogenous HMGB1 can promote MCF-7 cell proliferation and the occurrence of autophagy,as the same as the exocrine to conditioned medium of HMGB1 that increased the level of autophagy in MCF-7 cells and cell proliferation ability by up-regulated the expression amount of Beclin1,LC3 protein,decreased P62 protein expression(P < 0.01),and It depended on the activated condition of GPER in CAF cells.Conclusion: Small molecule compound G1 activates GPER of CAF in tumor microenvironment,,increases the secretion of cytokine HMGB1,and thus induces MCF-7 cells autophagy which can protect MCF-7 cells and promot them growth.These above effects may play an important role in the progress of breast cancer.