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

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。

Abstract:

[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- κ B pathway, enhances the EMT process of breast cancer cells and promotes bone metastasis of breast cancer.