Regulatory roles of miRNAs 16, 133a, and 223 on osteoclastic bone destruction caused by breast cancer metastasis.

Abstract

Osteolytic bone metastasis leads to skeletal-related events, resulting in a decline in the patient activities and survival; therefore, it is important to understand the mechanism underlying bone metastasis. Recent studies have suggested that microRNAs (miRNAs or miRs) are involved in osteoclast differentiation and/or osteolytic bone metastasis; however, the roles of miRNAs have not been elucidated. In the present study, the roles of miRNAs in bone destruction caused by breast cancer metastasis were investigated in vitro and in vivo. miR-16, miR-133a and miR-223 were transfected into a human breast cancer cell line, MDA-MB-231. The expression of osteolytic factors in conditioned medium (miR-CM) collected from the culture of transfected cells was assessed. To evaluate the effects of miRNAs on osteoclast differentiation and activities, tartrate-resistant acid phosphatase (TRAP) staining and bone resorptive assays were performed in osteoclasts following miR-CM treatment. To create in vivo bone metastasis models for histological and morphometric evaluation, miRNA-transfected MDA-MB-231 cells were transplanted into the proximal tibia of nude mice. Expression of osteolytic factors, including receptor activator for nuclear factor-κB ligand (RANKL), interleukin (IL)-1β, IL-6, parathyroid hormone-related protein (PTHrP), and tumor necrosis factor (TNF), was increased in miR-16-CM, whereas it was decreased in both miR-133a-CM and miR-223-CM. TRAP staining and bone resorptive assays revealed that osteoclast function and activities were promoted by miR-16-CM treatment, whereas they were suppressed by miR-133a-CM and miR-223-CM. Consistent with in vitro findings, in vivo experiments revealed that the overexpression of miR-16 increased osteoclast activities and bone destruction in MDA-MB-231 cells, whereas the opposite results were observed in both miR-133a- and miR-223-transfected MDA-MB-231 cells. Our results indicated that miR-16 promoted osteoclast activities and bone destruction caused by breast cancer metastasis in the bone microenvironment, whereas miR-133a and miR-223 suppressed them. These miRNAs could be potential biomarkers and therapeutic targets for breast cancer bone metastasis.