The Effects Of KISS1/GPR54System In Rat Osteosarcoma Cell Line UMR‐106and Orthotopic Model

Objectives1. To establish a in vivo rat's osteosarcoma model of "orthotopic transplantation–lung metastasis", so as to develop a laboratory foundation for the study on theeffects of KISS1/GPR54in rat with osteosarcoma;2. To determine the expression level of KISS1/GPR54system in the rat's bone tissueand the osteosarcoma model;3. To investigate the effect of Kisspeptin-10(Kp-10) on the cell migratory ability andinvasive ability of UMR-106;4. To construct the recombinant KISS1lentivirus, which was then used to transfectthe UMR-106, so as to establish UMR-106cell line, in which exogenous KISS1gene steadily expressed;5. To explore the potential impact and probable mechanism of KISS1transfection inrat's osteosarcoma.Methods1The barium suspension was used to measure the volume of rats' tibia bone marrowcavity. Appropriate amount UMR-106cell suspension was injected into the bonemarrow cavity. The orthotopic tumor growth and lung metastasis were assessed.In addition, the expressions of ALP (Alkaline Phosphatase) and OPN(Osteopontin) in the cell, orthotopic tumor and lung metastatic nodes weredetected by RT-PCR.2By employing RT-PCR and Western Blot, the expressions of KISS1and GPR54inthe rats' bone tissue, UMR-106and the orthotopic tumor tissue of nude mousemodels were detected with respect to mRNA level and protein level. 3. Three dosages of Kp-10(50nM,100nM and200nM) were added into to theUMR-106mono-layer, before wound healing assay and Transwell assay wereconducted respectively.4. The recombinant KISS1lentivirus was constructed for transfecting UMR-106. Byemploying the fluorescence assay method, the Multiplicities of Infection (MOI)was determined; by Western Blot, the expression of kisspeptin in transfected cellwas analyzed.5In vitro experiments: wound healing assay and Transwell assay were conducted toassess the variations on migratory and invasive ability of3cell lines (UMR-106cell, UMR-Vector and UMR-KISS1) respectively. Western Blot analysis was usedto determine the respective variations on the expressions of MMPs and TIMPs inthe3cell lines. In vivo experiments: the volumes of the orthotopic tumorsinduced by the transplantations of3cell lines were observed respectively, and thenumbers of the lung metastatic nodes were counted.Results1. The rat's osteosarcoma model of "orthotopic transplantation–lung metastasis" wassuccessfully established. The expressions of both ALP and OPN were observed inthe cell, orthotopic tumor and lung metastatic nodes.2. The expressions of KISS1were not detected in UMR-106and the orthotopic tumor.The expression of KISS1was relatively lower in bone tissue than in liver tissue.The expressions of GPR54were detected in all of the pre-mentioned tissues.3. After treating the UMR-106with Kp-10, both migratory distance and the numberof invaded cell significantly decreased (P<0.05).4. The recombinant KISS1lentivirus was successfully constructed and transfectedinto the UMR-106. The optimal value of MOI was200. After transfection, theexpression of KISS1in UMR-KISS1increased significantly and sustained formore than40days.5. In vitro experiments: in transfected UMR-KISS1cells, both migratory distance and the number of invaded cell significantly decreased, the expressions of bothMMP-2and MMP-9decreased, while the expressions of both TIMP-1andTIMP-2increased. In vivo experiment: in UMR-KISS1group, the volume oforthotopic tumor in nude mouse model reduced and the number of lungmetastastic nodes decreased.Conclusions1The orthotopic transplant in the rats' tibia bone marrow cavity by UMR-106couldcause lung metastasis, and the clinical features of osteosarcoma were retained.The surface between anterior tuberosity of tibia and medial edge of proximal tibiais the favorable site for transplantation.2. Lack of KISS1expression may be one of the reasons cause the metastasis ofosteosarcoma. The endogenous GPR54in osteosarcoma could be a potentialreceptor for gene therapy.3. Kp-10could inhibit the migration and invasion of UMR-106, and the inhibitoryeffect may depend on the dosage of Kp-10.4. The recombinant KISS1lentivirus could effectively transfect the UMR-106. In thetransfected UMR-KISS1cell line, the exogenous KISS1gene could steadilyexpression at a high level.5. The KISS1tranfection could inhibit the migration and invasion of osteosarcomacells in virto, and play an anti-metastasis role in the osteosarcoma nude mousemodels in vivo. The probable mechanism may be the up-regulation of TIMPs andits resultant decrease of MMPs level.