RANKL诱导内皮间质化促进乳腺癌骨转移  

RANKL induces endothelial mesenchymation to promote bone metastasis of breast cancer

作　　者：李勇爱 孟通 宋滇文 杨正峰 岳智颖 尹华斌 LI Yong-ai;MENG Tong;SONG Dian-wen;YANG Zheng-feng;YUE Zhi-ying;YIN Hua-bin(Department of Spinal Surgery,First People's Hospital,School of Medicine,Shanghai JiaoTong University,Shanghai 201620,China;Clinical Transformation Research Institute,First People's Hospital,School of Medicine,Shanghai JiaoTong University,Shanghai 201620,China)

机构地区：[1]上海交通大学医学院附属第一人民医院脊柱外科,上海201620 [2]上海交通大学医学院附属第一人民医院临床转化研究院,上海201620

出　　处：《中国矫形外科杂志》2024年第13期1215-1221,共7页Orthopedic Journal of China

基　　金：上海市自然科学基金项目(编号:22ZR1450500);上海市卫生健康委员会科研项目(编号:20234Z0020)。

摘　　要：[目的]探讨核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)在乳腺癌骨转移中的作用及其机制。[方法]MDA-MB-231,BT549和MCF-7乳腺癌细胞体外培养,加入终浓度为200 ng/ml RANKL(RANKL组),或同体积双蒸水(Ctrl组),行transwell、单细胞钙成像、PCR和western blot检测。分离小鼠骨髓细胞(bone marrow stromal cells,BMSCs),在培养其中加入乳腺癌细胞上清,行TRAP、ALP和von Kossa染色。[结果]RANKL在MDA-MB-231细胞[(1.7±0.1)个vs(1.0±0.1)个,P=0.002]、BT549细胞[(1.6±0.1)个vs(1.0±0.1)个,P=0.027]的相对迁移数显著高于Ctrl组。RANKL-200组MCF7[阳性数/总数(%),58/127(45.7)vs 3/118(2.5),P<0.001]和BT549细胞[阳性数/总数(%),51/224(22.8)vs 31/228(13.6),P=0.011]显著高于Ctrl组。PCR检测,RANKL-200组中MDA-MB-231细胞中的E-cadherin显著低于Ctrl组,N-cadherin、vimentin表达量显著高于Ctrl组。Western blot显示,N-cadherin相对表达水平依次为RANKL-500组>RANKL-200组>Ctrl组(P<0.05);而E-cad-herin蛋白表达水平依次为RANKL-500组<RANKL-200组<Ctrl组(P<0.05)。在小鼠骨髓细胞体外试验中,加入含RANKL上清组的TRAP染色阳性细胞数[(451.3±15.0)个vs(174.3±9.2)个,P<0.001]显著高于Ctrl组,而ALP染色和Von Kossa染色差异无统计学意义(P>0.05)。[结论]RANKL诱导的钙信号经NF-κB通路增强乳腺癌细胞EMT过程进而促进乳腺癌的骨转移。[Objective]To explore the role and mechanism of receptor activator of nuclear factor-κB ligand(RANKL)in bone metastasis of breast cancer.[Methods]The MDA-MB-231,BT549 and MCF-7 breast cancer cells were cultured in vitro,with the final concentration of 200ng/ml RANKL(RANKL group),or the same volume of double distilled water(control,Ctrl group).Transwell,single-cell calcium imaging(SCCI),PCR,and western blot assays were performed.The bone marrow stem cells(BMSCs)of mice were isolated,and cultured with the supernatant of breast cancer cells,which contained RANKL.TRAP,ALP and von Kossa staining were performed.[Results]The relative migration cell numbers of RANKL added MDA-MB-231 cells[(1.7±0.1)vs(1.0±0.1),P=0.002]and BT549 cells[(1.6±0.1)vs(1.0±0.1),P=0.027]were significantly higher than those in the Ctrl group.The RANKL-200 group showed significantly higher levels of MCF-7[positive/total(%),58/127(45.7)vs 3/118(2.5),P<0.001]and BT549 cells[positive/total(%),51/224(22.8)vs 31/228(13.6),P=0.011]compared to the Ctrl group.PCR detection showed that E-cadherin in MDA-MB-231 cells of the RANKL-200 group was significantly lower than that in the Ctrl group,while the expression levels of N-cadherin and vimentin in the former were significantly higher than those in the latter.Western blot showed that the relative expression levels of N-cadherin were in the following order:RANKL-500 group>RANKL-200 group>Ctrl group(P<0.05),whereas the expression levels of E-cadherin protein were in the order of RANKL-500 group<RANKL-200 group<Ctrl group.In the in vitro experiment of mouse bone marrow cells,the number of TRAP stained positive cells in the group containing RANKL supernatant was significantly higher than that in the Ctrl group[(451.3±15.0)vs(174.3±9.2),P<0.001],while there was no statistically significant difference between ALP staining and Von Kossa staining(P>0.05).[Conclusion]RANKL induced calcium signaling is mediated by NF-κB pathway,enhances the EMT process of breast cancer cells and promotes bone metastasis of brea

关 键 词：RANKL 乳腺癌骨转移 钙振荡 EMT NF-ΚB信号通路 

分 类 号：R318[医药卫生—生物医学工程]