Abstract
Extracellular matrix glycoprotein Reelin is associated with tumor metastasis and prognosis in various malignancies. However, its effects on multiple myeloma (MM) are not fully understood. Here, we investigated the regulatory effects of Reelin on MM and its underlying pathogenic mechanisms. Lentivirus plasmid containing short hairpin RNA targeting Reelin (LV3-Reln) was transfected into SP2/0 cells to knockdown Reelin expression. Flow cytometry assay analyzed cell cycle and apoptosis while Transwell assay evaluated invasiveness. BALB/c mice were inoculated with LV3-Reln-transfected SP2/0 cells to establish MM model. Primary myeloma cells and osteoblasts/osteoclast were isolated from tumor tissue and limb long bones respectively. ELISA examined serum biomarkers and immunohistochemistry detected immunoglobulin light chain expression. Morphological changes and osteoclast/osteoblast differentiation were observed by histological staining. mRNA and proteins expression were determined by qPCR and WB. In vitro studies showed that Reelin depletion regulated osteolysis and osteogenesis balance, cell cycle, invasiveness, and apoptosis in SP2/0 cells. In LV3-Reln mice, tumor growth and invasiveness were suppressed, meanwhile, reduced osteoclast activation and enhanced osteoblast activity were observed. Reelin knockdown alleviated extramedullary morbidity and inhibited spleen immune cell apoptosis by down-regulating CDK5, IL-10, and Cyto-C expression. Furthermore, reduced Reelin expression restrained osteoclast differentiation while promoted osteogenesis in the bone of LV3-Reln mice. This was further supported by down-regulation of osteolytic specific mRNAs and proteins (Trap, Mmp9, Ctsk, Clcn7) and up-regulation of osteogenic specific ones (COL-1, Runx2, β-Catenin). Reelin exerted important impacts on myeloma development through rebalancing osteolysis and osteogenesis, thus might be a potential therapeutic target for MM.
Highlights
Multiple myeloma (MM) is a neoplastic plasma-cell disorder characterized by hypercalcemia, anemia, renal insufficiency and bone disease, among which bony lesion developed in almost 80% of newly-diagnosed patients [1]
We further explored whether Reelin activity Bone disease characterized by osteolysis and osteopenia was a defining clinical manifestation of MM [23]
After observing Reelin’s depletion alters myeloma cell proliferation, apoptosis, and role in suppressing MM tumor growth, we investigated the invasiveness, influence OC and OB activity, and rebalance changes of key proteins involved in bone lesions
Summary
Multiple myeloma (MM) is a neoplastic plasma-cell disorder characterized by hypercalcemia, anemia, renal insufficiency and bone disease, among which bony lesion developed in almost 80% of newly-diagnosed patients [1]. After observing Reelin’s depletion alters myeloma cell proliferation, apoptosis, and role in suppressing MM tumor growth, we investigated the invasiveness, influence OC and OB activity, and rebalance changes of key proteins involved in bone lesions. As levels at day 14, 28, and 42 to explore whether Reelin can inhibit demonstrated in Fig. 5A with Trap staining, it is evident that the size the production of this monoclonal protein, suppressing the of OC isolated from LV3-Reln mice were smaller than those from pathologic effects of MM.
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