中文摘要：

目的：探讨小分子化合物G1活化微环境肿瘤相关成纤维细胞（cancerassociated fibroblast,CAF）中G蛋白偶联雌激素受体（G protein-coupled estrogen receptor,GPER）对外分泌细胞因子高迁移率族蛋白1（high mobility group box-1 protein,HMGB1）的影响,以及对乳腺癌MCF-7细胞自噬和增殖能力的影响。方法：构建靶向干扰GPER基因的GPER-shRNA慢病毒,稳定转染至CAF中;分别采用实时荧光定量PCR法及蛋白质印迹法检测阴性对照慢病毒感染组（CAF-shNC组）与GPER-shRNA慢病毒感染组（CAFshGPER组）中GPER mRNA和蛋白的表达情况。利用GPER特异性激动剂G1（1μmol/L）处理以上2组细胞后,分别得到G1＋CAF-shNC组与G1＋CAF-shGPER组细胞;应用实时荧光定量PCR法、蛋白质印迹法及ELISA法检测上述4组细胞中细胞因子HMGB1 mRNA和蛋白的表达水平,以及条件培养液中HMGB1的分泌量。收集以上各组细胞的条件培养液处理MCF-7细胞后,应用蛋白质印迹法检测肿瘤细胞自噬蛋白标志物Beclin1、p62及LC3的表达情况,CCK-8法检测对MCF-7细胞增殖能力的影响。结果：携带有GPER-shRNA的慢病毒稳定转入CAF后,GPER mRNA及蛋白的表达水平被明显抑制（P值均〈0.01）。GPER特异性激动剂G1可明显上调CAF-shNC组细胞中细胞因子HMGB mRNA及蛋白的表达水平,进一步上调条件培养液中HMGB1蛋白的分泌量（P值均〈0.05）。在G1＋CAF-shGPER组,中以上效应则被明显抑制。外分泌至条件培养液中的HMGB1可通过上调Beclin1和LC3蛋白以及降低p62蛋白的表达水平增强乳腺癌MCF-7细胞的自噬水平及其增殖能力（P值均〈0.01）。结论：小分子化合物G1通过活化微环境CAF中GPER,增加细胞因子HMGB1的分泌量,进而诱导乳腺癌MCF-7细胞发生自噬,保护MCF-7细胞并促进其生长。

英文摘要：

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 was detected by of four grou Beclinl, p62 proliferation secretion of HMGB1 protein in the conditioned medium of four groups enzyme- ps was co and LC3 of MCF-7 Ilecte nked immunosorbent assay （ELI d and used to treat MCF-7 cells. proteins in MCF-7 cells was detected cells were detected by CCK-8 assay SA）. The conditioned medium Then the expression levels of by Western blotting, while theproliferation 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