Study The Mechanism Of CD147/EMMPRIN On The Activation Of Osteoclasts In Metastatic Bone Tumors

BACKGROUND AND PURPOSES:Bone is a common target sites of metastatic tumor, many malignant tumor will developbone metastasis. The incidence of bone metastases was significantly higher than primarybone tumors. Prostate cancer, breast cancer and lung cancer are common tumors whichprone to metastasis to bone. Osteolytic is more common in Bone destruction caused bybone metastases, it can cause a sequence of serious complications including osteoporosis,pathologic fracture,stubborn pain, hypercalcemia,spinal cord compression syndrome, ect.The5-years survival rate of patients will significantly drop. It would seriously affect thehealth of patients and the patients’ quality of life.The series of cytokines in the cancer-bone microenvironment involved the process oftumor-bone metastasis, which regulate the interaction between tumor cells, bone cells andbone matrix, resulting in promoted tumor growth, and bone destruction. osteoclasts play aimportant role in bone destruction and absorption, tumor cells could promote osteoclastformation and activation by a variety of ways and a variety of factors,and mediatedosteolytic bone destruction occurred. Therefore, osteoclasts as a key target to research andtreat tumor-bone destruction, we will be in-depth study.CD147is also known as extracellular matrix metalloprotease inducer (EMMPRIN),which is correlate with tumor invasion, metastasis and prognosis. Recent studies haveshown that CD147could induced monocyte/macrophage activation by regulating thesecretion of MMPs, and participated in the destruction of alveolar bon,articular cartilageand articular bone in periodontitis and rheumatoid arthritis. Another study found thatinhibited the expression of CD147in breast cancer cells, the process of bone metastases andbone destruction of breast cancer had been effectively reduced. Therefore, we speculate thatCD147may be involve in the process of tumor-bone metastases and bone destructiondirectly or indirectly. Our previous study had found that CD147could be highly expressed on the surface of osteoclasts, and involved in regulating the formation and activation ofosteoclasts in vitro experimental.In view of the CD147involved in tumor biologicalbehavior by regulating the secretion of MMPs in stromal cells，and MMPs is an importantregulatory factor in the activation of OCs. So, we speculate that the tumor cells couldregulating the activation of osteoclasts by CD147, resulting in bone destruction occurs, it’smechanism may be associated with CD147regulate the secretion of MMPs.Therefore, this study will establish the model of induces peripheral blood mononuclearcells (PBMCs) differentiation into osteoclasts in vitro, and research the effect of CD147onthe synthesis, secretion and activation of MMPs during the differentiation of osteoclasts,explore the regulatory mechanism and modalities of CD147regulated the activation ofosteoclasts; Detective the expression of CD147and the relationship between CD147andthe activation of osteoclasts in bone tumor, to preliminary verify that the conclusions of theprevious study in vitro experiments.so that we could have a prefect understanding ofCD147involved in the processes of tumor-bone destruction, And provide experimentalreference and theoretical basis to the next in-depth study for our research group.METHODS:1．Separated mononuclear cells from peripheral blood by ficoll density gradientcentrifugation；purificated the separated mononuclear cells by stick wall.2．Induced PBMCs differentiation into OCs by exogenous M-CSF protein and RANKLprotein；TRAP staining and bone resorption experimental identificated the generated cells,and to detected the bone resorption of the generated cells；3．Cells were separately divided into two groups[control group (normal induction group)and the protein group] in the first part of the experiments and two groups [control group(normal induction group) and the blocking group] in the second part of the experiments.4．Observed cells morphology and TRAP stained on the12th day; Observed boneabsorption lacunes on bone chips by toluidine blue dye after cells cultured in bone chips for30days;5．Detected the mRNA levels of CD147、MMP-2and MMP-9by Real-time PCRduring the differentiation of Ocs at24h and48h time points, then analysis their correlation;6．Detected the protein levels of CD147、MMP-2and MMP-9by Enzyme linkedimmunosorbent assay (ELISA) during the differentiation of Ocs at24h and48h time points; 7．Detected the activity enzyme levels of MMP-2and MMP-9by Gelatin zymographyduring the differentiation of Ocs on24h and48h time points;8．Detected the protein levels of CD147、MMP-2and MMP-9by SP immunohistochemical in metastatic bone tumor；9．Detected the mRNA levels of CD147、MMP-2and MMP-9by RT-PCR inmetastatic bone tumor；10．Observed and identified OCs in metastatic bone tumor by HE and TRAP staining;11. Detected the protein levels of CD147by SP immunohistochemical in OCs ofmetastatic bone tumor；12．SPSS17.0statistical software for data analysis, statistical significance was set at P <0.05RESULTS:1．Separated mononuclear cells from peripheral blood by ficoll density gradientcentrifugation and purificated the separated mononuclear cells by stick wall, the numberand purificated of mononuclear cells meet our vitro experiments requirement;2. Induced PBMCs differentiation into OCs by exogenous M-CSF protein and RANKLprotein，the generated Cells were TRAP staining (+) and with bone resorption capacity；3. The number of TRAP(+) cells of protein group was significantly more than controlgroup`s On the12th day, and the size of osteoclasts was bigger; The number of TRAP(+)cells of blocking group was significantly less than control group`s On the12th day, nomultinucleated cell formation; The number of absorption lacunes of protein group andcontrol group were significantly more than the control group, and the protein group couldformed vast stretches of absorption lacunes, but the blocking group could not formedabsorption lacunes;4．The mRNA levels of CD147、MMP-2and MMP-9of OPCs in protein group at24h,48h time points were higher than corresponding control group`s(P＜0.05)；The mRNAlevels of CD147、MMP-2and MMP-9in blocking group at24h,48h time points werelower than corresponding control group`s(P＜0.05); Moreover, the mRNA levels ofMMP-2and MMP-9was significantly positively correlated to the mRNA levels of CD147in both research[（R=0.818�'�R=0.758，P<0.05）,（R=0.525�'�R=0.552，P<0.05）]；5. The protein levels of CD147、MMP-2and MMP-9in protein group at24h,48h timepoints were higher than corresponding control group`s(P＜0.05) 6. The enzyme activity levels of MMP-2and MMP-9was significant differencesbetween the blocking group and control group（P＜0.05），and the activity enzyme levelsof MMP-2and MMP-9in blocking group at24h，48h time points were lower thancorresponding control group`s(P＜0.05)；7. The protein expression levels of CD147, MMP-2and MMP-9in the metastatictumor group were significantly higher than benign bone tumor group’s (P <0.05).8. The mRNA levels of CD147、MMP-2and MMP-9in metastatic tumor group werehigher than benign bone tumor group’s (P＜0.05)；Moreover, the mRNA levels of MMP-2and MMP-9was significantly positively correlated to the mRNA levels of CD147（R=0.795�'�R=0.956，P＜0.05）；9. The number of multinucleated giant cells were located in the interface between thebone tissue and tumor tissue, Identified these multinucleated giant cells were osteoclasts byTRAP staining;10. The positive expression of CD147protein in the cytoplasm and membrane ofosteoclasts in metastatic bone tumor.CONCLUSIONS:1．Induced PBMCs differentiation into OCs by exogenous M-CSF protein and RANKLprotein，the generated Cells were TRAP staining (+) and with bone resorption capacity, thismodel is more reliable to meet our vitro experiments requirement;2．Exogenous CD147protein could enhanced the mRNA expression of CD147,MMP-2and MMP-9, and it also enhances the secretion levels of MMP-2, MMP-9duringthe process of osteoclast differentiation. it’s suggested that exogenous CD147was relatedto the synthesis and secretion of MMPs during the process of osteoclast differentiation;3. CD147mAb could inhibited the mRNA expression of CD147、MMP-2and MMP-9in OPCs, and it also inhibits the enzyme activity levels of MMP-2and MMP-9during theprocess of osteoclast differentiation, it’s suggested that endogenous CD147was related tothe the enzyme activity levels of MMPs during the process of osteoclast differentiation;4. The protein and mRNA levels of CD147, MMP-2and MMP-9were higherexpression in the metastatic tumor, it’s suggested that CD147, MMP-2and MMP-9may berelated to the tumor-bone metastases and bone destruction；5. The protein levels of CD147were higher expression in OCs, it’s suggested that CD147may be related to the activation of OCs in metastatic tumor.