Feng Qi, Wang Donglai, Feng Jiangang, Guo Peng, Geng Cuizhi
Department of Orthopedics, The Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, China.
Breast Disease Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, China.
Transl Cancer Res. 2020 Apr;9(4):2482-2491. doi: 10.21037/tcr.2020.03.17.
Denosumab is an inhibitor of receptor activator of NF-κB ligand (RANKL), which inhibits bone metastasis (BM) in breast cancer (BC), but does not completely control cancer cell BM in some BC patients. This study was designed to study whether denosumab inhibits human BC cells (MCF-7) cell line-induced spontaneous osteoclastogenesis via RANKL/metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/miR-124 axis.
We established a co-culture system of MCF-7-induced spontaneous osteoclastogenesis in RAW 264.7 cells, and denosumab is added into the co-culture system to inhibit RAW 264.7 cell differentiation into osteoclasts. Real-time PCR (RT-PCR), immunofluorescence and western blotting analysis were used to detect gene expression, while tartrate-resistant acid phosphatase (TRAP) staining was used to assess osteoclast formation.
Denosumab inhibits MCF-7 cell line-induced spontaneous osteoclastogenesis, and the inhibition of denosumab was found to be more pronounced after MALAT1 downregulation and miR-124 overexpression. However, MALAT1 knockdown or miR-124 overexpression did not alter RANKL protein expression. Moreover, the dual luciferase gene reporter system showed that miR-124 targeted the inhibition of MALAT1, while si-MALAT1 upregulated miR-124 expression. miR-124-mimics were able to decrease the expression of Rab27a, IL-11, activated T-cell nuclear factor 1 (NFATc1) and TARP protein.
Denosumab inhibits MALAT1 expression by inhibiting RANKL, thereby upregulating miR-124 expression, which ultimately inhibits MCF-7 cell line-induced pseudo osteoclastogenesis.
地诺单抗是一种核因子κB受体活化因子配体（RANKL）抑制剂，可抑制乳腺癌（BC）的骨转移（BM），但在部分BC患者中并不能完全控制癌细胞的BM。本研究旨在探讨地诺单抗是否通过RANKL/转移相关肺腺癌转录本1（MALAT1）/miR-124轴抑制人BC细胞（MCF-7）细胞系诱导的自发性破骨细胞生成。
我们建立了MCF-7诱导RAW 264.7细胞自发性破骨细胞生成的共培养体系，并在共培养体系中加入地诺单抗以抑制RAW 264.7细胞分化为破骨细胞。采用实时荧光定量聚合酶链反应（RT-PCR）、免疫荧光和蛋白质印迹分析检测基因表达，同时用抗酒石酸酸性磷酸酶（TRAP）染色评估破骨细胞形成。
地诺单抗抑制MCF-7细胞系诱导的自发性破骨细胞生成，并且发现下调MALAT1和过表达miR-124后，地诺单抗的抑制作用更明显。然而，敲低MALAT1或过表达miR-124并未改变RANKL蛋白表达。此外，双荧光素酶基因报告系统显示miR-124靶向抑制MALAT1，而小干扰RNA-MALAT1（si-MALAT1）上调miR-124表达。miR-124模拟物能够降低Rab27a、白细胞介素11（IL-11）、活化T细胞核因子1（NFATc1）和破骨细胞相关受体蛋白（TARP）的表达。
地诺单抗通过抑制RANKL抑制MALAT1表达，从而上调miR-124表达，最终抑制MCF-7细胞系诱导的假性破骨细胞生成。
