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Fig. 5 | Breast Cancer Research

Fig. 5

From: Metastatic breast cancer cells overexpress and secrete miR-218 to regulate type I collagen deposition by osteoblasts

Fig. 5

CM from miR-218-overexpressing cells inhibits type I collagen processing in differentiated MC3T3. a Western blot analyses of type I collagen and Timp3 in the CM from differentiated MC3T3 treated with indicated EV-depleted CM for 16 days. CM producing cell lines were transfected, PBS washed 48 h after transfection and then incubated with serum-free medium overnight before CM collection and EV depletion by ultracentrifugation. Osteoblast differentiation factors were then added into collected CM, which was used to treated MC3T3 cells and replaced every 3–4 days. Cellular β-actin was used as control. b Relative RNA level of Timp3 normalized to 18S in CM-treated differentiated MC3T3 at day 18. ***P < 0.001. c Relative RNA level of Timp3 in CM-treated differentiated MC3T3 at day 18. Anti-inhibin βA (1 ng/ml) antibody was added to CM as indicated. ***P < 0.001. d Western blot analyses of Timp3 in the CM from differentiated MC3T3 treated with indicated CM with or without anti-inhibin α antibody for 16 days. Cellular β-actin was used as control. e Proposed model of bone niche adaptation mediated by miR-218 through direct secretion and targeting of type I procollagen in osteoblasts (1) or regulation of inhibin βA whose secretion in turn blocks procollagen processing by osteoblasts (2)

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