GPER-mediated HMGB1 exocrine in cancer-associated fibroblasts promotes autophagy and proliferation of breast cancer MCF-7 cells / 肿瘤

ABSTRACT

Objective: To investigate the effects of high mobility group box-1 protein (HMGB1) exocrine promoted by G protein-coupled estrogen receptor (GPER) in cancer-associated fibroblast (CAF) combined with the small molecule compound G1 on the autophagy and proliferation of breast cancer MCF-7 cells. Methods: The GPER-shRNA lentiviral vector specifically interfering GPER gene expression was constructed and used to infect CAF (CAF-shGPER), while the CAF infected with a negative control lentivirus was used as the control (CAF-shNC). The expression levels of GPER mRNA and protein in CAF-shNC and CAF-shGPER cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The GPER-specific agonist G1 was used to treat the CAF-shNC and CAF-shGPER cells, respectively. Then the expression levels of HMGB1 mRNA and protein in CAF-shNC, CAF-shGPER, CAF-shNC+G1 and CAF-shGPER+G1 cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The secretion of HMGB1 protein in the conditioned medium of four groups was detected by enzyme-linked immunosorbent assay (ELISA). The conditioned medium of four groups was collected and used to treat MCF-7 cells. Then the expression levels of Beclin1, p62 and LC3 proteins in MCF-7 cells were detected by Western blotting, while the proliferation of MCF-7 cells was detected by CCK-8 assay. Results: After the lentivirus carrying GPER-shRNA was stably infected into CAF, the expressions of GPER mRNA and protein were significantly inhibited (both P<0.01). The expressions of HMGB1 mRNA and protein in CAF-shNC cells were significantly up-regulated by GPER specific agonist G1 (both P<0.05), while the secretion of HMGB1 was increased in the conditioned medium (P<0.05); However, the above effects of G1 were opposite in CAF-shGPER cells. Furthermore, the exocrine HMGB1 in conditioned medium up-regulated the expressions of Beclin1 and LC3 proteins (both P<0.01), down-regulated the expression of p62 protein (P<0.01), and increased the autophagy and proliferation abilities of MCF-7 cells (both P<0.01). Conclusion: The small molecule compound G1 can promote the expression of GPER in CAF in tumor microenvironment and increase the secretion of cytokine HMGB1, thus induce the autophagy of MCF-7 cells and promote the growth of MCF-7 cells.