肿瘤相关成纤维细胞中GPER介导HMGB1外分泌促进乳腺癌MCF-7细胞自噬及增殖  

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

作　　者：郭林英[1] 余腾骅 张光君[1] 尹恒[1] 柳满然[3] 涂刚[1] 

机构地区：[1]重庆医科大学附属第一医院内分泌乳腺外科,重庆400016 [2]江西省肿瘤医院乳腺外科,江西南昌330029 [3]重庆医科大学临床检验诊断学教育部重点实验室,重庆400016

出　　处：《肿瘤》2017年第5期448-456,共9页Tumor

基　　金：国家自然科学基金面上项目(编号:81372398)~~

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。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

关 键 词：乳腺肿瘤 G蛋白偶联雌激素受体 HMGB1蛋白 细胞自噬 细胞增殖 

分 类 号：R737.9[医药卫生—肿瘤]