| Background and objectivesGiant cell tumor (GCT) of bone is one of the most common primary bone tumors, and accounts for 6% of all the bone tumors. GCT of bone has a predisposition for people aged 20-40 years, typically occurs in the metaphyseal regions of long bones. Although defined as a kind of benign tumor, GCT often exhibits strong local aggressiveness. Osteolysis is an essential feature of GCT, which is closely related to severe pain and might cause pathological fracture. GCT of bone is insensitive to radiotherapy and chemotherapy, and surgical resection is the main treatment for this disease. With the progress of the surgical technique, en-bloc resection is widely used in the treatment of GCT, however, the overall postoperative recurrence rate of GCT can be up to 41.7%. Because of the local aggressiveness and high recurrence rate, GCT is also presently recognized as a type of borderline or potential malignant bone tumor. Osteolytic destruction and abnormal proliferation are the typical pathological features of GCT, which could influence the life quality and overall survival of the patients with GCT. However, the mechanism of GCT is still not fully understood.GCT derives from bone marrow mesenchymal cell, and is mainly made up of three types of cells:osteoclast-like multinucleated giant cells, spindle-shaped stromal cells, and mononuclear cells. Previous studies considered that multinucleated giant cells were the main effector cell and tumor component of GCT. But recent studies show that spindle-like stromal cells of GCT (GCTSC) are the only tumor component in GCT with infinite proliferation ability, and play a key role in osteolytic destruction. GCTSC originates from bone marrow mesenchymal cell (BMSC). Unlike BMSC, it can not only secrete a great deal of osteolysis-related cytokines to induce multinucleated osteoclasts formation and osteolysis, but also maintain abnormal proliferation of GCT. Therefore, exploring how GCTSC have the ability of abnormal proliferation and excessive secretion of osteolysis-related factors is crucial to reveal the occurrence and osteolytic destruction of GCT, and also helps to improve the prevention and treatment of GCT.Matrix metalloproteinase 13 (MMP-13) is an important member of the matrix metalloproteinase family, and also an important collagenase in vivo. MMP-13 can effectively degrade aggrecan in cartilage, and type Ⅰ, Ⅱ, Ⅲ collagen in cell surface. MMP-13 plays an important role in many physiological and pathological processes invivo, such as connective tissue disease, wound healing, angiogenesis, tumor invasion and metastasis etc. Previous studies have found that MMP-13 was highly expressed in GCT and could attribute to osteolysis formation.Parathyroid hormone related peptide (PTHrP), which is a polypeptide hormone secreted by the parathyroid gland, shares the same N terminal structure with parathyroid hormone and plays a role in vivo through a combination with parathyroid hormone 1 receptor. PTHrP has also been confirmed to be highly expressed in GCT and play a vital role in GCT. It could promote the formation of osteolytic lesion by up-regulation of RANKL (NF-κB ligand), MMP-13 and IL-8, and meanwhile enhance proliferation ability of GCTSC by inhibition of apoptosis.The Runt transcription factor -2 (Runx2) is an important member of Runt family, and plays an vital role in differentiation and formation of bone cells. Previous studies have found that there is an up-regulation of Runx2 in GCT, which can affect the biological behavior of GCT by regulating the expression of a series of factors.MicroRNAs (miRNA) are small, evolutionarily conserved noncoding RNA molecules with an average length of 22 nt in vivo and that act as posttranslational regulators by combining to the mRNA 3’UTR region of downstream target genes and suppress their translation process. Bioinformatics analysis of the human genome sequence revealed that more than 1/3 of human gene might be regulated by microRNAs. The expression and effect of microRNAs in tumors have become the hotspot and focus of foreign and domestic research, but their function in GCT is still not clear. MicroRNA 126-5p is an intronic miRNA located in the epidermal growth factor-like domain 7 (EGFL7) gene and is widely expressed in vivo. MiR-126-5p plays an extensive and important role in the human body, and a number of studies have shown that it stimulates endothelial cell proliferation and involves in cardiovascular diseases such as atherosclerosis, myocardial infarction, and so on. MiR-126-5p has been identified as a tumor suppressor in many tumors such as prostate cancer, melanoma, breast cancer and non-small cell lung cancer, its role in GCT has not been reported.Research contents and methodsWe collected typical tumor specimens from patients with GCT who were surgical treated at the Changzheng Hospital Affiliated to the Second Military Medical University from July 2011 to June 2012. The GCT specimens, together with cancellous bone samples were detected by microRNA chips to found microRNAs with differential expression. Multiple target gene prediction databases and bioinformatics analyses were used to find microRNAs with differential expression and their potential target genes.qRT-PCR, in situ hybridization, and fluorescence immunoassay detection of GCT specimen and cancellous bone amples were used to test the expression of miR-126-5p in GCT. Meanwhile, qRT PCR, western blot, and ELISA were used to analyze the potential target gene expression in GCT, while HE and immunohistochemical staining were used to further confirm the expression of miR-126-5p and its potential target gene in GCT. By building the luciferase activity report system and point mutation of binding sites, we tried to confirm the relationship of miR-126-5p and its potential target genes. We transfected GCTSC and MG63 with miR-126-5p mimic and miR-126-5p antagonist to further define the regulation of miR-126-5p on its targets.We constructed a GCTSC cell of miR-126-5p overexpression (OE-miR-126 GCTSC) by Talen, and tested it with qRT-PCR, western blot and ELISA analyses. Bone marrow mononuclear cell and OE-miR-126 GCTSC were co-cultured to observe osteoclast differentiation and formation of multinucleated giant cells in order to make clear the role of miR-126-5p on osteoclast differentiation. Dentin slices was co-cultured with OE-miR-126 GCTSC to see the role of miR-126-5p in osteolysis formation. Cell count, CCK8 experiments, and cell cycle detection were used to analyze the role of miR-126-5p on GCTSC proliferation.For further analyze the role of miR-126-5p on osteolytic microenvironment and GCTSC proliferation in vivo, we builted a series of animal models for validation. First we builded a ovary osteoporosis mice model (OVX mice model) and used miR-126-5p intervention to verify the role of miR-126-5p in osteolytic microenvironment; Then we used chicken embryo experiments to analyze the effect of miR-126-5p on GCTSC proliferation; Finally, we constructed a miR-126-5p specific knockout mice by Talen technology and comprehensively observe the impact of miR-126-5p on the process of growth and development in mice.Results and conclusionIn this study, we found the following results:1. MicroRNA chip analysis and qRT-PCR test of tissue samples found that miR-126-5p was obviously downregulated inGCT.2. Bioinformatics analysis revealed that miR-126-5p might paly a vital role in GCT. Fourthermore, MMP-13, PTHrP and Runx2 might be the potential target genes of miR-126-5p.3. qRT-PCR, western blot and ELISA analyses revealed that MMP-13, PTHrP, Runx2, RANKL, and IL-8 were expressed in high level in GCT samples; while HE and immunohistochemical staining results further suggested the above factors were secreted by GCTSC.4. Luciferase experiments have established that miR-126-5p could directly combine with MMP-13, PTHrP and Runx2 mRNA 3’UTR region, and inhibit their protein expression. Transfection experiments further confirmed that miR-126-5p mainly affect the protein expression levels of MMP-13, PTHrP, and Runx2.5. We constructed a miR-126-5poverexpression GCTSC with Talen technique, and the cell could increase the miR-126-5p expression by 20 times. 6. Co-culture of OE-miR-126 GCTSC with bone marrow mononuclear cell could restraine osteoclast differentiation and decrease multinucleated giant cell formation.7. MiR-126-5p overexpression decreased bone destruction in dentin slices experiment.8. GCTSC proliferation experiment suggested miR-126-5p overexpression could significantly suppressed GCTSC proliferation.9. In the OVX mice model, miR-126-5p overexpression could restrain the bone loss process in OVX mice, while low expression of miR-126-5p accelerated bone loss and aggravate bone destruction in OVX mice.10. Chicken embryo allantois capsule experiment found that miR-126-5p overexpression inhibited GCTSC proliferation and GCT occurrence;11. We successfully constructed 12 F1 generation of miR-126-5p specific knockout mice model by useing Talen technology. They were used to observe the impact of miR-126-5p on the process of growth and development in mice.Through the above results, we demonstrated that the miR-126-5p played an important role in the occurrence and biological behavior of GCT. It could influence GCTSC proliferation and the formation of osteolytic microenvironment by direct regulation of MMP-13, PTHrP and Runx2. Meanwhile we found that miR-126-5p could also indirectly regulate the expression of RANKL and IL-8, and there is a more complex control relation between MMP-13, PTHrP, and Runx2. Therefore, miR-126-5p plays a extremely complex role in GCT. |
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