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

发布时间：2018-08-07 21:34

【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。
[Abstract]:Aim: to investigate the effect of G protein-coupled estrogen receptor (G protein-coupled estrogen receptor) on exocrine cytokine high mobility group protein 1 (high mobility group box-1 protein 1 (HMGB1) in cancerassociated fibroblast associated fibroblasts (cancerassociated fibroblast caf) activated by small molecule G 1. And the effect on autophagy and proliferation of breast cancer MCF-7 cells. Methods: GPER-shRNA lentivirus targeting GPER gene was constructed and stably transfected into CAF. The expression of GPER mRNA and protein in lentiviral infection group (CAF-shNC group) and GPER-shRNA lentivirus infection group (CAFshGPER group) were detected by real-time fluorescence quantitative PCR and Western blotting respectively. After treated with GPER specific agonist G1 (1 渭 mol/L), the cells of G1 CAF-shNC group and G1 CAF-shGPER group were obtained, respectively. The expression of cytokine HMGB1 mRNA and protein and the secretion of HMGB1 in conditioned medium were detected by Western blot and ELISA. After MCF-7 cells were treated with conditioned medium, the expression of Beclin1p62 and LC3 in tumor cells was detected by Western blotting. The effect of CCK-8 on the proliferation of MCF-7 cells was detected by CCK-8 method. Results: the expression of GPER mRNA and protein in lentivirus carrying GPER-shRNA was significantly inhibited (P = 0. 01). GPER specific agonist G1 could significantly up-regulate the expression of cytokine HMGB mRNA and protein in the cells of CAF-shNC group. The secretion of HMGB1 protein in conditioned medium was further up-regulated (P < 0. 05). In G 1 CAF-shGPER group, the above effects were significantly inhibited. HMGB1 secreted into conditioned medium could enhance the autophagy level and proliferation ability of breast cancer MCF-7 cells by up-regulating Beclin1 and LC3 protein and decreasing the expression of p62 protein (P < 0.01). Conclusion: small molecule compound G1 increases the secretion of cytokine HMGB1 by activating GPERin microenvironment CAF, and then induces autophagy, protects MCF-7 cells and promotes the growth of MCF-7 cells in breast cancer.
【作者单位】：重庆医科大学附属第一医院内分泌乳腺外科;江西省肿瘤医院乳腺外科;重庆医科大学临床检验诊断学教育部重点实验室;
【基金】：国家自然科学基金面上项目(编号:81372398)~~
【分类号】：R737.9

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