MicroRNA-106b Regulates Osteolysis By Targeting RANKL In Giant Cell Tumor Of Bone

Giant cell tumor (GCT) of bone, which accounts for approximately6%of all primarybone tumors, is characterized by extensive bone resorption, leading to regional pain andthe predisposition to pathologic fractures. Although considered as non-cancerous tumor,GCT of bone is known for its potential to recur following treatment. Histologically GCTconsists of three major cell types: the multinucleated osteoclast-like giant cells, themonocytic round-shaped macrophage-like cells, and the spindle-shaped, fibroblast-likestromal cells. Giant cell formation is thought to occur in a similar manner asosteoclastogenesis, where osteoblasts express the receptor activator of nuclear factor-kBligand (RANKL), which stimulates its receptor, RANK, on osteoclast precursor cells andinitiates their fusion into osteoclasts. Proliferating GCT stromal cells are believed to be theneoplastic component of GCT which are known to express RANKL and are thought tostimulate giant cell formation from the RANK expressing monocytic cells.RANKL, a member of the TNF ligand superfamily are essential for osteoclastogenesisin vivo. It is considered that RANKL drives osteoclast precursor cells to form activemultinucleated osteoclasts and is necessary for osteoclast differentiation, survival, andactivation. In addition, the up-regulation of RANKL also plays an important role in otherdisease which related to osteolysis, such as osteoporosis and osteofibrous dysplasia, aswell as bone metastases. Recent findings have revealed a number of transcription factorsthat regulate RANKL expression. However, as another vital processe for the final proteaseactivity, post-transcriptional regulation of RANKL has rarely been reported.MicroRNAs (miRNAs) are a class of abundant, approximately22nucleotidenon-coding RNAs that mediate post-transcriptional regulation of target mRNAs. Bybinding to the3’-untranslated regions (3’UTRs) of specific mRNAs, miRNAs represstranslation or degrade target mRNAs. Bioinformatics have predicted that miRNAs have thecapacity to regulate one third of all mammalian genes, and numerous studies have linkedaberrant miRNA expression to pathological conditions such as cancer. Despite this, thefunctions of miRNAs in GCT induced osteolysis still remain poorly understood. Given thesimilar forming process of giant cell in GCT and osteoclast, miRNAs that regulateosteoclastogenesis may play a key role in GCT of bone. Emerging evidence has revealed ageneral necessity for miRNAs in osteoclastogenesis. Nonetheless, the function of miRNAsrelating to RANKL, which is pivotal for osteoclastogenesis, still remains unclear. Mir-106b has been confirmed to upregulate in many malignant tumors in a role ofpromoting tumor cell proliferation. However, recent researches revealed that miR-106bwas downregulated in some bone metastases including breast cancer and renal cellcarcinoma compared with the primary tumor. Though some proteins involved in osteolysishave been reported to be the targets of miR-106b, there is still no direct report aboutmiR-106b and bone resorption.In this study, microRNA expressions in GCT tissues and normal bones were measuredby miRNA microarray assay and were determined by qRT-PCR. Luciferase assay wasperformed to validate the potential target of miR-106b. Overexpressed miR-106b(OE-106b) stromal cells were constructed by using TALE nucleases and the effect ofOE-106b was detected. OVX mouse model was chosen for its osteoclastic bone resorption.Each mouse was injected with agomiR-106b or antagomiR-106b or their controls. Then itwas detected the levels of target proteins, the activity of osteoclasts and the degree of boneresorption.We verified the downregulation of miR-106b in GCT of bone and futher provide aconclusive explanation for the essential role of miR-106b in osteoclast differentiation andbone resorption by:(a) identifying RANKL as one direct target of miR-106b;(b)demonstrating an inhibition of osteoclastogenesis and osteolysis induced by OE-miR-106bcells; and (c) revealing that high levels of miR-106b reduced bone resorption andosteoclast activity while low levels of miR-106b promoted them in vivo. Our findingsindicate that miR-106b negatively regulates RANKL and further inhibitsosteoclastogenesis and osteolysis. This may help the diagnosis and therapeutic of bonetumors or other diseases related to bone resorption.