The target gene investigation in osteoblats (OBs) treated with mmu-miR-1963 enriched in EVs from OCs with prostate cancer (PCa) cells [target_EV-miR-1963]

Bone metastatic lesions of PCa contain areas of bone destruction and formation that are directed by OCs and osteoblasts (OBs), respectively. OC-derived EVs are reported to maintain normal bone homeostasis; however, there is no study investigating the role of EVs from OCs in the presence of PCa. We identified mmu-miR-1963 enriched in EVs from OCs co-cultured with several PCa cell lines. NGS analyses were performed to investigate predicted target genes of EV-miR-1963 in OBs. Overall design: To investigate the role of EVs derived from OCs with PCa cells, we cultured murine monocytic cell line RAW 264.7, which has potential to differentiate into OC-like cells, with metastatic PCa cell lines and controls. We prepared different types of OCs and EVs from these cells ; OCs normally differentiated from RAW264.7 cells (OC cells), OC cells co-cultured with normal prostate epithelial cells: PNT2 cells (OCN cells), osteolytic PCa cells: PC3M cells (OCP cells), and osteoblastic PCa cells; C4-2B cells (OCC cells). OC differentiation was induced in the presence of RANKL. EVs were isolated from culture supernatant using ultracentrifugation. As a result, we identified mmu-miR-1963 as an enriched microRNA (miRNA) in OCP and OCC EVs. We prepared total RNAs extracted from OCP/OCC/OCN EV treated OBs and mmu-miR-1963/NC mimic transfected OBs and then NGS analyses were performed to investigate predicted target genes of EV-miR-1963 enriched in OCP and OCC EVs in mineralizing OBs (mOBs). Total RNAs are extracted 24h after the treatment in the presence of ascorbic acid and ß-glycerophosphate.