Correlation Of Autophagy Gene Beclin 1 To Tumorigenesis And Development Of Osteosarcoma

Background:The osteosarcoma is the most common malignant tumor of bone.It occurs mostly in teenagers,with high malignancy,early metastasis and poor prognosis.At present for which the common treatment,such as surgical operation,chemotherapy,is suboptimal and the prognosis of patients remains dismal.Therefore,novel therapeutic strategies are constantly being pursued.Various candidates for osteosarcoma therapy include new chemotherapeutic drugs,differentiating agents,vaccines,monoclonal antibodies,and approached using dendritic cell.With the development of technology,gene therapy has become a new treatment method for those patients who have lost the opportunity for operation.In case of cancer an abnormally increased cellular lifespan as a consequence of reduced apoptosis is ideal to favor the insurgence of genetic mutations,as well as to shield transformed cells from death induced by chemotherapy or radiotherapy,and to promote their survival at distant sites.Therefore,it is not surprising that manipulation of autophagy has emerged as a new therapeutic strategy to help eliminate cancer cells.Autophagic cell death is a type of programmed cell death different from apoptosis, caspase-independent.Some studies have showed that autophagy suppression may result in malignant transformation,and the inactivation of autophagy gene Beclinl induces malignancy,which is a candidate tumor suppressing-gene.So we believe that our study in the expression of Beclin 1 in the osteosarcomar is an exceedingly valuable investigation. The gene of Beclin 1 might be a more directly objective gene for gene therapy on osteosarcomar.This study has great significance in finding a new treatment strategy and the pathogenesis of osteosarcomar by the result of our investigation on the role of Beclin 1 in tumorigenesis and development of osteosarcomar.Therefore we have conducted the following three part of experimental study. The First Part The different expression of Becl in 1 in human osteosarcomar cell line MG63 and human osteoblast cell line hFOB1.19Objection To identify the different expression of Beclin 1 between the human osteosarcomar cell line MG63 and human osteoblast cell line hFOB 1.19 and to investigate the role of Beclin lin the tumorigenesis and development of osteosarcomar.Method Real time-polymerase chain reaction(RT-PCR) and Western blot were used for detecting the expression of mRNA and protein of Beclin 1 Result The expression of mRNA and protein of Beclinl in human osteosarcomar cell line MG63.is lower obviously than that in the human osteoblast cell line hFOB1.19;Conclusion Beclinl exression is down-regulated in human osteosarcomar cell line MG63,which may relate to tumorigenesis and development of osteosarcomar.The Second Part Construction of recombinant Becl in 1 tumor autophagy gene eukaryotic expression plasmidObjective To construct recombination eukaryotic expression plasmid carrying human Beclinl cDNA,transfect and overexpress in the osteosarcomar cell for gene therapy of osteosarcomar.Method A eukaryotic expression plasmid for pEGFP/Beclinl was constructed by use of recombinant DNA technique and was transfected into human osteosarcomar cell line MG63 by using lipofectamine 2000.The mRNA and protein expression of Beclinl were detected by the RT-PCR and Western blot method.Result The recombinant eukaryotic expression plasmid was demonstrated by restriction endonuclease mapping and sequencing from the company of Takara in Dalian.The Beclinl gene transfected human osteosarcomar cell line MG63 showed having mRNA expression of Beclinl Conclusion The recombinant plasmid was constructed corrected and the mRNA and the protein of Beclinl gene could be expressed in human osteosarcomar cell line MG63 which establish the base for gene therapy of osteosarcomarThe Third Part Growth suppression of human osteosarcomar cell line MG63 by transfer of wild Beclin 1 geneObjective To investigate whether Beclinl gene transfer and overexpression could inhibit the in vitro growth and the cells'sensitivity to MTX of human osteosarcomar cell line MG63.Method Eukaryotic expression vector pFGFP/Beclinl was transfected into human osteosarcomar cell line MG63 by using lipofectamine 2000,constructing with the pEGFP groups,and osteosarcomar cell groups only.The effect Beclinl overexpression on the proliferation and chemotherapy(MTX) result of human osteosarcomar cell line MG63 was evaluated by MTT assay.Cell apoptosis was measured by flow cytometry(FCM). MDC staining was used to study the condition of autophagy in cells.Result The inhibition rate was significantly higher in pEGFP/Beclinl cells than in controlgroups.At 72h after transfection,the apoptosis rate was significantly higher in pEGFP/Beclinl cells,same to the cells which was dealed with MTX and autophagy in transfected cells.Conclusion Beclinl overexpression can inhibit proliferation and promote apoptosis and autophagy of human osteosarcomar cell line MG63,which also could increase the cells' sensitivity to MTX.