MicroRNAs and their functional targets in osteosarcoma and non-tumor bone cells

Abstract

The pulmonary metastasis of osteosarcoma (OS) occurs commonly, which resulted from anoikis resistant (AR) of tumor cells as reported by previous studies, but the exact roles of AR in osteosarcoma were not fully studied. Our previous investigations showed fatty acid synthase (FASN) was relating to clinical features of patients with OS. In this study, we aim to explore the functions of FASN in the AR OS cells in vitro and in vivo and study the downstream effectors of FASN. In the present study, established cell model was used to study the AR. Results showed that AR promoted cell proliferation and migration as determined by Colony formation assay and Transwell assay. Additionally, AR assisted tumor growth in vivo. In the AR cells, the expression of FASN was higher. Thus, lentiviruses to silence or overexpress FASN in four cell lines were constructed to study functions of FASN. Silence of FASN reduced cell colonies and migration while overexpression of FASN increased colonies and migration in suspended cells. Loss of functions of FASN induced cell apoptosis in suspended OS cells whilst gain of function of FASN suppressed apoptosis as determined by flow cytometry. In vivo study showed that inhibition of FASN decreased pulmonary metastasis of OS. Finally, we conducted microRNAs (miRNAs) microarray to study the miRNAs regulating FASN in the OS cells. Results showed that miR-15a could downregulate FASN in OS cells. These studies could potentially contribute to the treatment of osteosarcoma. I also demonstrate matrix metallopeptidase 9 targeted by hsa-miR-494 promotes silybin-inhibited osteosarcoma. These molecules could be potentially used in translational medicine. Wnt signaling pathways are essential for bone formation. Previous studies showed that Wnt signaling pathways were regulated by miR-375. Thus, we aim to explore whether miR-375 could affect osteogenesis. In the present study, we investigated the roles of miR-375 and its downstream targets. Firstly, we revealed that miR-375-3p negatively modulated osteogenesis by suppressing positive regulators of osteogenesis and promoting negative regulators of osteogenesis. In addition, the results of TUNEL cell apoptosis assay showed that miR-375-3p induced MC3T3-E1 cell apoptosis. Secondly, miR-375-3p targeted low-density lipoprotein receptor-related protein 5 (LRP5) and β-catenin as determined by luciferase activity assay, and it decreased the expression levels of LRP5 and β-catenin. Thirdly, the decline of protein levels of β-catenin was determined by immunocytochemistry and immunofluorescence. Finally, silence of LRP5 in osteoblast precursor cells resulted in diminished cell viability and cell proliferation as detected by WST-1-based colorimetric assay. The mRNA levels of important negative modulators relating to osteogenesis increased after the functions of LRP5 were blocked in mice. Last but not least, the expression levels of LRP5 increased during the osteogenesis of MC3T3-E1, while the levels of β-catenin decreased in bone tissues from osteoporotic patients with vertebral compression fractures. In conclusion, we revealed miR-375-3p negatively regulated osteogenesis by targeting LRP5 and β-catenin. In addition, loss of functions of LRP5 damaged bone formation in vivo. We also showed that strontium inhibited osteoclastogenesis by enhancing LRP6 and β-catenin-mediated osteoprotegerin (OPG) targeted by miR-181d-5p.