The Effect Of The Mitochondrial Fusion Protein2Expression On Proliferation And Apoptosis Of Osteosarcoma And It’s Molecular Mechanismy

Objective: Mitofusin-2is a important member of mitofusin family, which has a lowexpression level in many cancer cells including MG63osteosarcoma cell. The objective ofthis study is to observe the impact of Mfn2on the proliferation and apoptosis ofosteosarcoma cell in vivo and in vitro experiments, to further reveal the function of thisgene on proliferation and apoptosis of cancer cells, which may lay a solidtheory foundation for the treatment of osteosarcoma.Methods:(1) To analysis the expression level of mfn2in osteosarcoma andpara-carcinoma tissue through immunohistochemical technique; to detect the mRNA andprotein expression levels of mfn2in normal and MG63osteosarcoma cell lines.(2) Toconstruct a pIRES-flag-Mfn2recombinant plasmid, and then transfect the recombinantplasmid into MG63cell, and then further screen out clones those expression flag-mfn2.(3)To detect the proliferation and apoptosis of MG63osteosarcoma cell lines after theexpression level of mfn2has up-regulation: to detect the proliferation through MTT; toobserve the cell morphology through inverted fluorescence microscope or electronmicroscopy; to detect the cell cycle through flow cytometry and to detect apoptosis relatedprotein such as PARP, caspase3; DNA fragmentation detection.(4) To culture the cellsthose express flag-Mfn2and Harvest the cell lysates, to purify the flag-Mfn2proteinthrough Immunoprecipitation and make mass spectrum analysis, and then discovery theinteractional protein that proliferation and apoptosis related; to research and analysis thepossible mechanism that the mfn2slows down the proliferation and accelerates theapoptosis of osteosarcoma cell.Results:(1) the expression level of mfn2in osteosarcoma is significant lower than thenormal cell and tissue, and the expression level of mfn2in osteosarcoma is lower thanpara-carcinoma tissue.(2) successfully constructed a human pIRES-flag-Mfn2recombinant plasmid, then transfect the recombinant plasmid into MG63cell, we havedetected the up-regulation of the expression of mfn2through RT-PCR and Western blot.(3)the clone formation assay show the number of cells has reduced and the proliferation hassuppressed after the transfection. The MTT shows the reduction of proliferation rate andthe elevated of the inhibition rate of the cells those mfn2is overexpression. flowcytometry anslysis the cell cycle: the rise of the G1phase cells and the reduce of the SandG2phase cells have found in those mfn2is overexpression. The cell cycle has been arrested in G1phase; detect the expression of the apoptosis related protein such as PARP:Cleaved PARP in the cells those mfn2is overexpression, induced the apoptosis.(4).Immunoprecipitation and mass spectrum analysis have found the interactional proliferationand apoptosis related protein:such as aspase-14, PARP and14-3-3protein.Conclusion:This study make a preliminary suggestion that the mfn2may be anoveling tumor suppressor gene. Through the observation of the impact on theproliferation and apoptosis of cancer cell in vivo and in vitro experiments. to further revealthe function of this gene on proliferation and apoptosis of cancer cells, which may lay asolid theory foundation for the treatment of osteosarcoma.