miRNA-106b失活可通过上调MMP2表达参与乳腺癌骨转移

倪小健; 张宏伟; 朱玮

摘要: 目的：探讨基质金属蛋白酶2(matrix metalloproteinase 2，MMP2)及其调控子has-miR-106b (miRNA-106b/miR-106b)在乳腺癌骨转移中的作用及机制。方法：采用定量PCR、免疫组织化学染色、Western 印迹法测定乳腺癌骨转移患者MMP2、 miR-106b的表达，并分析MMP2与乳腺癌骨转移患者临床特征的关系。细胞迁移和侵袭实验观察MMP2、miR-106b表达变化体外对乳腺癌细胞迁移和侵袭的影响。荧光素酶报告基因检测MMP2和miR-106b的靶向关系。Western 印迹验证受调控的下游信号通路。结果：MMP2在侵袭能力较强的细胞如SUM1315-bo中表达较高，在侵袭能力较弱的乳腺癌细胞如MCF-7中表达较低；而miR-106b的表达与之相反（P<0.05）。与未发生骨转移的乳腺癌患者相比，MMP2蛋白在乳腺癌骨转移患者原位肿瘤标本中表达较高；miR-106b的表达与之相反（P<0.05）。MMP2促进乳腺癌细胞的迁移和侵袭，miR-106b反之（P<0.05）。miR-106b可下调 MMP2 的表达，进而影响下游调控因子p-ERK/ERK的表达（P<0.05）。在SUM1315-bo中下调 MMP2 基因后，其培养基培养的骨髓间充质干细胞(human bone marrow-derived mesenchymal stem cell, HMSC)定向分化为成骨细胞过程中，细胞核因子κB受体活化因子配体(RANKL)/骨保护素(osteoprotegerin, OPG)轴失衡，即 OPG含量增加、 RANKL含量减少，导致破骨细胞分化减少（P<0.05）。结论：MMP2过表达是乳腺癌骨转移的危险因素，这可能与miR-106b失活有关；MMP2 可能通过调节ERK信号通路而促进乳腺癌溶骨性骨转移；miR-106b-MMP2-ERK信号通路是乳腺癌骨转移潜在的预测因子及治疗靶标。

Abstract: Objective：To investigate the function and mechanism of matrix metalloproteinase 2 (MMP2) and micro-RNA-106b (miR-106b) in the breast cancer bone metastasis. Methods：The expressions of MMP2 and miR-106b were measured by immunohistochemistry, RT-PCR, and Western blotting in breast cancer bone metastasis tissue samples, and the relationship between MMP2 and clinical features of breast cancer bone metastasis patients was analyzed. The influence of MMP2 and miR-106b on migration and invasion of breast cancer cells in vitro was analyzed. The targeting relationship between miR-106b and MMP2 was confirmed by luciferase target assay. Western blotting was used to verify the regulated downstream signaling pathways. Results： The expression of MMP2 was higher in invasive cells, such as SUM1315-bo, but lower in less invasive breast cancer cells, such as MCF-7. However, the expression of miR-106b was opposite. Compared with breast cancer patients without bone metastasis, the expression of MMP2 protein in breast cancer patients with bone metastasis was higher, while the expression of miR-106b was the opposite. MMP2 promoted the migration and invasion of breast cancer cells, while miR-106b did the opposite (P<0.05). MiR-106b downregulated the expression of MMP2, and then affected the expression of downstream regulatory factor p-ERK/ERK. After downregulation of MMP2 gene in SUM1315-bo, the cultured human bone marrow-derived mesenchymal stem cell (HMSC) differentiated into osteoblasts, in which process receptor activator for nuclear factor-κB ligand (RANKL) / osteoprotegerin (OPG) axis lost balance-the content of OPG increased and the content of RANKL decreased, resulting in reduced osteoclast differentiation (P<0.05). Conclusions：Overexpression of MMP2 may be one of the risk factors for bone metastasis in breast cancer, which may be related to the inactivation of miR-106b. MMP2 may promote the osteolytic bone metastasis of breast cancer by regulating the ERK signaling pathway. MiR-106b-MMP2-ERK signaling pathway has the potential to be a predictive factor and a therapeutic target for bone metastasis in breast cancer.