The Expression Of BRMS1 In Osteosarcoma And The Research Of Its Mechanism To Inhibit The Metastasis Of Osteosarcoma

PartⅠ: The expression of BRMS1 in osteosarcoma and its significanceObjective:to research the expression of BRMS1 in osteosarcoma and the relationship between the expression of BRMS1 and the metastasis potential of osteosarcoma .Methods: with the reference of the expression ofβ-actin ,the expression of BRMS1 was determined by reverse transcription-polymerase chain reaction in 70 osteosarcoma tissues and the normal adjacent tissue. 15 patients of giant cell tumor are also analysed at the same time .within the 70 patients of osteosarcoma 10 patients had tumor metastasis but the other 60 patients had no metastasis .Results: expression of BRMS1 was different in osteosarcoma tissue and the normal adjacent tissue,it is 0.378±0.046 and 0.918±0.044 ,the different was statistic and the expression of BRMS1 had no relation with patients age ,the size of tumor but it was related to the ability of metastasis of osteosarcoma .the average expression level of giant cell tumor is 0.718±0.045 which had statistic difference compared with that of osteosarcoma but the giant cell tumor and the normal tissue had no statistic difference .the group of having metastasis and the group having no metastasisi of the patients of osteosarcoam also had statistic difference .Conclusions: the expression of BRMS1 in the osteosarcoma is lower than that of the normal tissue and the expression of it in giant cell tumor is higher than that of osteosarcoma so it is related to the potential of metastasis of the osteosarcoma .PartⅡ: The construction and identification of eukaryotic expression vector of BRMS1Objective :To construct the eukaryotic expression plasmid of pCDNA3.1/myc-BRMS1 and prepare the study of its expression and function in osteosarcoma cells.Methods :Total RNA was isolated from muscle cells , and the full-length coding sequence of BRMS1 was constructed by standard reverse transcriptional PCR method. Then the fragment was linked with pcDNA3.1/myc-His(+)A and then inserted into the shuttle vector T. Transformation of E.coli DH-5αwith recombinant plasmids and identification of bacterial colonies containing recombinant plasmids by LB-agar plate containing 100μg/ml of ampicillin were conducted, and recombinant plasmids were extracted and purified. All sequences amplified by PCR were confirmed by complete sequencing. Correct sequences were cloned into the pcDNA3.1/myc-His(+)A vector. pcDNA3.1/myc-BRMS1 was transfected into cell. The BRMS1 expression was detected by RT-PCR and Western blot.Results: Recombinant pcDNA3.1/myc-BRMS1 was gained and it contained the correct recombinant BRMS1 sequences. The base sequence of recombined pcDNA3.1/myc-BRMS1 was in accordance with fragment sequence of human BRMS1cDNA and the full length pcDNA3.1/myc-His (+)A by agarose gel electrophoresis and gene sequence analysis .Conclusions :The pcDNA3.1/myc-BRMS1 plasmids can express the target gene in high level. The plasmid is constructed successfully and will be useful for further research.on the base of pcDNA3.1/myc-BRMS1 we can explore the influence of BRMS1 to the cell of osteosarcoma ,especially its influence to the ability of metastasis. PartⅢ: The transfection of eukaryotic expression vector of BRMS1 to MG-63 and identification of the characteristics of transfected cellsObjective: to transfect MG-63 cells with pcDNA3.1/myc-BRMS1 and study the effect of BRMS1 gene on the biological behaviour of osteosarcoma cells and to explore the mechanism of its suppression effect of the metastasis of osteosarcoma .Methods: Methods The eukaryotic expression vector pcDNA3.1/myc-BRMS1 containing CDS sequence of BRMS1 cDNA and empty plasmid vector pcDNA3.1/myc-His (+)A was transfected into MG-63 cells by lipofectamine respectively;after G418 selection ,transfected cells was cloned ; the morphology was observed by light microscope ; scanning electron microscope was used to observe the ultrastructure of cells; the expression of BRMS1 mRNA in cells before and after transfection was detected by RT-PCR ; the cellular proliferating ability was assessed by MTT; the change of the cell cycle and apoptosis was analyzed by flow cytometry, the ability of invasion and motility was assayed by modified Boyden Chamber;the change of GJIC was detected by SLDT (Scrape loading dye transfer, SLDT) .Results: After transfection ,compared to transfectants of MG-63 cells with empty plasmid vector and parental MG-63 cells. The change of morphology was not observed by light microscope, but ultrastructures of surface had obvious change; BRMS1 mRNA of transfected MG-63 cells was up-regulated significantly; the cellular proliferating ability did not alter (P>0.05); the differences were not observed for the change of the cell cycle,apoptosis (P>0.05);the ability of invasion in vitro was decreased significantly.,and motility was inhibited too(P<0.05); the gap junction intercellular communication (GJIC) was retored; the empty plasmid vector pcDNA3.1/myc-His (+)A did not alter the character of MG-63 cells.Conclusions: after transfection , BRMS1 mRNA of transfected cells was up-regulated significantly ,the reason that suppress metastasis were that①ultrastructure of cells had obvious change ,②the ability of in vitro invasion decreased significantly and the motility was inhibited too③the gap junction intercellular communication was restored .empty plasmid vector pcDNA3.1/myc-His(+)A did not alter the character of osteosarcoma cells .PartⅣ: The nude mice study of the tumor metastasis of transfected MG-63 cellsObjective: To establish nude mice model for osteosarcoma with spontaneous metastasis and experimental metastasis ,and to investigate the effect of suppressing osteosarcoma metastasis in nude mice by BRMS1 gene ,and to find a effective method to treat osteosarcoma metastasis .Methods: MG-63 cells with eukaryotic expression vector of BRMS1 and empty plasmid vector pcDNA3.1/myc-His(+)A were inoculated subcutaneously into tibia in nude mice and select with G418. nude mice mere divided into three groups ,three different suspensions of MG-63 cells were inoculated respectively to establish the model of spontaneous metastasis ,then other group of nude mice were divided into three groups ,three different suspensions of MG-63 cells were injected into the lateral tail vein respectively to establish the model of experimental metastasis ,the animals were housed under SPF condition ,after 5 weeks all mouse were sacrificed by cervical dislocation ,the lung and livers were obtained and examined by nectopsy and histopathology . all above date were analysed statistical to find the difference between these groups .Results: in the model of spontaneous metastasis , The mean weight of nude mice and mean diameters of nude mice were not different significantly in each groups , in the group of experimental metastasis ,the mean weight of nude mice were different significantly in each groups (A2/B2;A2/C2),the number of metastasis colonies to lungs was 3. 5±0.5,16.1±2.1 and 15.4±2.4 (sites/one mouse ) ;the number of metastasis colonies to liver was 0,3.5±1.2 and 4.2±0.5 (sites/one mouse ) there were significant difference to be found between the groups of experimental metastasis(A2/B2;A2/C2) and no statistic difference in the groups of experimental metastasis .the group of B2 and C2 did not differ statistically in the number ofmetastasis colonies to lungs or liver.Conclusions: BRMS1 gene can suppress the experimental metastasis in nude micebut it does not influence the primary tumor proliferation ,this test providedexperimental data and support to the effect of BRMS1 gene to the metastasis ofosteosarcoma . These give us a new way to treat the osteosarcoma especially to reducethe metastasis potential of osteosarcoma .