Study Of The Tumor Inhibitory Gene RECK On The Invasional Biologic Behavior Of Osteosarcoma

Part oneThe preliminary study on the expression of the tumorinhibitory gene RECK in the osteosarcoma tissues[Objective] To detect the the expression of RECK gene in osteosarcoma tissuesexplore its possible roles on the occurrence and metastasis of osteosarcoma.[Methods] The expression level of RECK protein in 38 osteosarcoma tissues and 38normal bone tissues were detected by immunohistochemistry.[Results] Of the 38 osteosarcomas examined,26 were stained weak or negative forRECK,whereas the strong RECK staining was observed in all 38 normal bone tissues.Theabsorbent optical density value of RECK protein in osteosarcoma tissues and normalbone tissuses were (0.31±0.07) and (0.58±0.08),respectively.The expression of RECK inosteosarcoma tissues was significantly lower than that in normal bone tissuse(P＜0.01).RECK protein expression was significant correlated with the pulmonary metastasis(P＜0.05,P=0.019),but had no correlation with age,sex,tumor size and pathologic grades.Ina comparison between the patients with RECK-positive tumors and patients withRECK-negative tumors,the survival time of the RECK-positive group was found to be 31months,whereas that of the RECK-negative group was 15 months,indicating theRECK-positive group had significantly better survival rate than the RECK-negative group(P＜0.05,P=0.032 by Log-rank test).[Conclusion] The abnoamal low expression of RECK may participate in osteosarcomainvasion and metastasis,and may be a new prognostic molecular marker for osteosarcoma and a new therapeutic target for tumor.Part twoThe study of transfecting RECK gene on MMP-2 activationand invasional ability of human osteosarcoma cell[Objective] To investigate effect of transfecting RECK gene on MMP-2 activation andinvasional ability of human osteosarcoma cell line MG-63.[Methods] The recombinant eukaryotic expression vector pcDNA3-RECK inserted bythe full length cDNA encoding human RECK gene was stably transfected into MG-63 byLipofectamine^TM 2000.RT-PCR,westem blot and flow cytometric analysis were used toassay RECK gene expression.MMP-2 activation ratio of the cell supematant and cellinvasional ability of MG-63 were analyzed by gelatinase zymography and matrigel invasionassay,respectively.The cytotoxicity of plasmid DNA transfection on MG-63 cells weredetermined by MTT assay and cell growth curve method.[Results] The stable and higher expression of RECK in mRNA and protein level weredetected in MG-63,respectively.Gelatinase zymography showed MMP-2 activation ratio intransfecting blank plasmid group and normal control group were (0.72±0.11),(0.80±0.14).Nosignificant difference was found between them.However,the MMP-2 activation ratio intransfecting recombinant plasmid group was (0.23±0.03),which was obviously less thanthose in transfecting blank plasmid group and normal control group(both P value＜0.01).Mrtrigel invasion assay showed cell number invading through Matrigel in transfecting blankplasmid group and normal control group were (28.18±2.15)and(30.25±2.23),respectively.Nosignificant difference was found between them.The cell number invading through Matrige intransfecting recombinant plasmid group was (14.20±1.26),which was dramatically decreasedcompared with those in transfecting blank plasmid group and normal control group(both Pvalue＜0.05).MTT showed no significant difference was found in transfecting recombinant plasmid group compared with transfecting blank plasmid group and normal control group incomparing cell growth curve (both P value＞0.05).[Conclusion] Over expression of RECK gene significantly inhibited MMP-2 activationand cell invasional ability of osteosarcoma cell MG-63,suggesting RECK may be a newtarget for osteosarcoma treatment.