MICA Protein Expression In Osteosarcomas And The Preliminary Study On The Role Of MMP9 In MICA Proteolytic Process

Osteosarcoma is the most common malignant bone tumor with poor prognosis in children and teenagers. Owing to the feature of its invasive growth, fast cell proliferation and high recurrence rate,osteosarcoma is hard to be removed completely by the currently available combined therapeutic approaches, such as surgical resection, postoperative radiotherapy, chemotherapy and immunotherapy etc. Its high recurrence rate seriously affects the life quality and overall survival of the patients, and the clinical prognosis is poor. As the knowledge of tumor biology and molecular genetics increased, it has been shown that the development of osteosarcoma, just the same as the tumors in the other sites of the body, results from the activation of proto-oncogenes and inactivation of tumor suppressor genes, and involves multiple genetic and molecular alterations. Therefore, seeking more genes associated with the osteosarcomas, a comprehensive understanding of the molecular pathology of osteosarcomas, and optimizing treatment strategies and developing novel therapeutic approaches for osteosarcomas on this basis, have become an important research project for osteosarcomas. It can be said that as one of the hot spots in the study of osteosarcomas, the gene therapy for correcting the aberration of genetic events in osteosarcomas has been the forefront focus in medical research field. In the present study, we focused on the MICA protein expression in osteosarcomas and the role of MMP9 in MICA proteolytic process in osteosarcomas. The study was divided into 2 parts.(1) the MICA expression were studied in the 39 cases benign bone tumors and 40 cases osteosarcomas by immunohistochemistry to estimate the different MICA expression in benign bone tumors and osteosarcom. Also the MICA expression of total protein and supernate protein in osteosarcoma cell line were detected by western-blot to explore the roal of MICA in osteosarcoma cell line.(2) the expressions of MMP2 and MMP9 of supernate protein in osteosarcoma cell line were studied in vitro by zymography. The MICA expression of total protein and supernate protein in osteosarcoma cell line were detected by western-blot again after adding inhibitorⅠ,MMP2/MMP9 inhibitor V to the cell culture to inhibit the activities of MMP2,MMP9 respectively to explore the roal of MMP2 and MMP9 in the MICA proteolytic process.Result:(1) Immunohistochemical expression of MICA was examined in 40 malignant osteosarcomas and 39 benign bone tumors. Tumor cells from osteosarcoma displayed up-regulated expression of MICA compared with adjacent bone tissue. Among 40 osteosarcomas,31 (77.5%) were positive for MICA. Expression levels were scored as-,+,++ and +++ and were found to be 9/40 (22.5%),12/40 (30%),11/40 (27.5%), and 8/40 (20%), respectively. Among 39 benign tumors, 25/39 (64.1%),7/39 (18%),5/39 (12.8%), and 2/39 (5.1%) cases were scored as -,+,++ and +++, respectively. There was a significant difference in MICA expression between the osteosarcomas and benign bone tumors. However,there were no significant differences among the osteosarcomas in different stages. MICA expression was also detected in the stroma of all positive expressed tumors, indicating that MICA shedding is a very common event in the different stages of osteosarcomas. Both cell lysates and concentrated conditioned culture media were analyzed by immunoblot assays using anti-MICA antibody, which recognizes the N-terminal portion of MICA. MICA protein was found not only in supernatants but also in cell lysates. The theoretical molecular weight of MICA is 45KDa. There are 8 N-glycosylation sites located within the extracellular domain. To detect the MICA cleavage site, supernatants were treated with PNGaseF followed by immunoblotting. The MICA signal was detected in the region of 30KDa. Using ImageQuant TL 7.0 Image Analysis Software, we determined the size of sMICA to be 33KDa, indicating that sMICA might be shed from theα3 domain of MICA.(2) We detected high MMP9 activity in MNNG cells by gelatin zymography, and 5nM of MMP9 inhibitor I decreased sMICA release within 48 hours. The cells were then treated with the inhibitor using concentrations ranging from 5nM to 50nM for 48 hours. The inhibitor decreased sMICA release and increased the membrane-bound MICA in a dose-dependent manner. Next, we used the MMP2/MMP9 inhibitor V, which specifically inhibits the activity of MMP2 at 16nM. We also detected high MMP2 activity in MNNG cells. However,16nM of MMP2/MMP9 inhibitor V had no effect on sMICA release. These results suggest that MMP9, but not MMP2 might be involved in MICA proteolytic process.Conclusion:(1) Both osteosarcoma and benign bone tumors displayed MICA expression, and there was a significant difference in MICA expression between the osteosarcomas and benign bone tumors. However, there were no significant differences among the osteosarcomas in different stages. In osteosarcoma cell line sMICA might be shed from theα3 domain of MICA.(2) In osteoarcoma MMP2 has no effect on sMICA release,but MMP9 promotes the MICA proteolytic process even mediates MICA shedding,which plays a key roal for osteosarcoma immunoescape.(3) MMP9 may become molecular therapy target candidates in osteosarcoma, and inhibiting the MMP9 activities potentially prevent the osteosarcoma from immuno escaping.