The Research Of Correlations Between Osteopontin (OPN) Expression And Osteosarcoma Pulmonary Metastasis:in Vivo And In Vitro

Osteosarcoma is one of the most common primary malignant and aggressive tumors of bone, occurring often in adolescents and young adults, the most common forms of treatment is wide margin tumor resection, amputation, neo-adjuvant and adjuvant chemotherapy. However, over the past few decades, with all the advancement in surgical techniques and drug development the 5-year survival rate of 65%,60% respectively hasn’t changed. Greater than 30% of patients develop lung metastases despite aggressive combination chemotherapy and surgery, with pulmonary metastasis being the major common cause of death in Osteosarcoma patients. However, these patients are less likely to enroll on clinical trials due to late stage diagnosis, significant chemotherapy related toxicity. However, with advancement in technology and newer treatment strategy, such as angiogenesis drugs will pave path for future prognosis and treatment.OPN is an arginine-glycine-aspartate (RGD)-containing secretion adhesive glycoprotein that was first identified as a major sialoprotein in bone and subsequently found to be expressed in kidney, brain, macrophages, vascular smooth muscle cells and many cells of epithelial linings. OPN is an acidic glycoprotein with a molecular mass of w44 kDa. OPN protein has been associated with regulation of adhesion, migration and other biological behavior of cells by specific signaling pathways. Recent studies have reported that OPN protein could also have close association to malignant tumor invasion, metastasis and proliferation of many pathologic processes as well.1. OBJECTIVESTo observe the difference of OPN expression between mice lung metastases and primary tibia osteosarcoma. To verify weather silencing of OPN could promote cell invasion in osteosarcoma cells.2. METHODS2.1 The difference of OPN expression between tibia osteosarcoma and lung metastasis:in vivoThe luciferase gene was transfected in osteosarcoma K7M2 cells using trsanfection agent lipofectamine 2000. After successful tranfection, cells were selected in the presence of G418 in culture medium,in order to obtain stable transfected cell.The signal of luciferase gene was measured with IVIS Lumina XRMS Series Ⅲ imaging system. Transfected cell viability was measured using the Cell Counting Kit-8 (CCK-8). To construct a mouse tumor model,50ul of transfected K7M2 cells at density 5×108/ml were injected into right tibia marrow cavity of BALB/C mice. The luciferase signal was tested with IVIS Lumina XRMS Series Ⅲ imaging system every 3 days after injection. Once the lung metastasis signal was detected (8 weeks after injection), the mice were continued to rare for additional 4 weeks. Then the mice were sacrificed and lung tumor tissues were collected and cultured to obtain primary osteosarcoma cells. The above procedure was repeated for another two times, by subculturing the obtained pulmonary metastasis osteosarcoma cells and reinjecting in the new BALB/c mice. After the 2nd time mouse tumor models were sacrafised and the tibia tumors, lung metastatic tissues and normal tibia tissues were collected and used for further testing. Western blot was performed to compare the OPN expression among the three kinds of tissue samples. Immunohistochemical staining was used to verify the western blot result.2.2 The verification of the function of OPN protein:in vitroThree kinds specific siRNA of OPN gene (siRNA1,2,3) and none specific siRNA were synthesis in vitro and transfected into the above-obtained cells using Lipofectamine2000 reagent. The down regulated OPN expression was identified by real time PCR. Cell invasiveness was tested with Transwell cell invasion assay and scratch test in vitro.3.RESULTSThe transfected K7M2 osteosarcoma cells could stably express luciferase gene, without any changes in their cell viability. Western blot assay verified increased OPN protein expression in the lung metastases compared to that of tibial osteosarcoma (p<0.0001). The immunohistochemical staining of the primary osteosarcoma and lung metastasis demonstrated similar result as western blot. OPN expression was lowered in OPN silenced K7M2 cells by siRNA 1,2,3 in comparison to none specific siRNA and lowest in siRNA2 group using real time PCR (p<0.0001). Transwell cell invasion assay and scratch test showed OPN silenced cells possessed less invasiveness than normal cells.4. CONCLUSIONS(1) OPN protein is highly expressed at the osteosarcoma metastatic site (lungs) than that of primary osteosarcoma site (Tibia)(2) The over expression of OPN protein in human osteosarcoma cell line, positively correlates with cell invasiveness.