CaSR Induces Osteoclast Differentiation and Promotes Bone Metastasis in Lung Adenocarcinoma
CaSR Induces Osteoclast Differentiation and Promotes Bone Metastasis in Lung Adenocarcinoma
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CaSR 诱导破骨细胞分化并促进肺腺癌骨转移
DOI：
10.3389/fonc.2020.00305
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发表时间：
2020-03-25
影响因子：
4.7
通讯作者：
Yu, Jing
中科院分区：
文献类型：
--
作者：
Liu, Lian;Fan, Yichang;Yu, Jing
目的：探讨钙调素受体（CaSR）参与肺腺癌骨转移的机制。研究方法：应用免疫组化法检测120例肺腺癌骨转移灶中钙敏感受体（CaSR）的表达。以A549细胞为基础，构建CaSR过表达和敲低的稳定转染细胞系。通过western blot和qPCR验证CaSR的表达。采用胆囊收缩素-8（CCK-8）法和Transwell法检测A549细胞的增殖能力和迁移能力。Western blotting检测MMP 2、MMP 9、CaSR、NF-κB的表达。收集各细胞培养组的上清液作为条件共培养液，研究破骨细胞前体细胞和成骨细胞的诱导。采用Western blot和qPCR验证骨基质降解相关酶组织蛋白酶K和激素降钙素受体（CTR）以及成骨细胞诱导的破骨细胞成熟和分化酶核因子-κB配体受体激活剂（RANKL）、巨噬细胞集落刺激因子（M-CSF）、骨保护素（OPG）和PTHrP的表达。免疫荧光染色检测F-actin环形成和骨钙素表达。Western blot检测NF-κB的表达，证实NF-κB与CaSR之间存在调节关系。结果：肺癌组织中CaSR的表达明显高于癌旁组织和正常肺组织。有骨转移的肺癌组织中CaSR的表达高于无骨转移的肺癌组织。CaSR过表达可显著增强A549细胞的增殖和迁移能力。共培养液直接诱导破骨细胞前体细胞，骨基质降解相关酶的表达显著增加。成骨细胞明显受到抑制，成骨细胞诱导的破骨细胞成熟和分化酶明显下调。发现CaSR过表达时，NF-κB和PTHrP的表达增加。破骨细胞分化因子的表达也明显增加，直接诱导破骨细胞分化成熟。当CaSR被敲除时，这些结果被逆转。结论：CaSR可正向调节高转移潜能A549细胞NF-κB和PTHrP的表达，从而促进破骨细胞分化成熟，促进肺腺癌骨转移的发生和发展。
Objective: Explore the mechanism of CaSR's involvement in bone metastasis in lung adenocarcinoma. Methods: Immunohistochemistry (IHC) was used to detect the expression of calcium-sensing receptor (CaSR) in 120 cases of lung adenocarcinoma with bone metastasis. Stably transfected cell lines with CaSR overexpression and knockdown based on A549 cells were constructed. The expression of CaSR was verified by western blot and qPCR. The proliferation and migration abilities of A549 cells were tested using cholecystokinin-8 (CCK-8) and Transwell assays, respectively. Western blotting was used to detect the expression of matrix metalloproteinases MMP2, MMP9, CaSR, and NF-κB. The supernatant from each cell culture group was collected as a conditional co-culture solution to study the induction of osteoclast precursor cells and osteoblasts. Western blot and qPCR were used to validate the expression of bone matrix degradation-related enzymes cathepsin K and hormone calcitonin receptor (CTR) and osteoblast-induced osteoclast maturation and differentiation enzyme receptor activator of nuclear factor-κB ligand (RANKL), macrophage colony-stimulating factor (M-CSF), osteoprotegerin (OPG), and PTHrP. Immunofluorescent staining was used to detect F-actin ring formation and osteocalcin expression. Western blot results for NF-κB expression identified a regulatory relationship between NF-κB and CaSR. Results: CaSR expression in lung cancer tissues was significantly higher than that in adjacent and normal lung tissues. The expression of CaSR in lung cancer tissues with bone metastasis was higher than that in non-metastatic lung cancer tissues. The proliferation and migration ability of A549 cells increased significantly with overexpressed CaSR. The co-culture solution directly induced osteoclast precursor cells and the expression of bone matrix degradation-related enzymes significantly increased. Osteoblasts were significantly inhibited and osteoblast-induced osteoclast maturation and differentiation enzymes were significantly downregulated. It was found that the expression of NF-κB and PTHrP increased when CaSR was overexpressed. Osteoclast differentiation factor expression was also significantly increased, which directly induces osteoclast differentiation and maturation. These results were reversed when CaSR was knocked down. Conclusions: CaSR can positively regulate NF-κB and PTHrP expression in A549 cells with a high metastatic potential, thereby promoting osteoclast differentiation and maturation, and facilitating the occurrence and development of bone metastasis in lung adenocarcinoma.