Study On ZFX Expression In Osteosarcoma Cell Lines And Functional Regulation On Malignant Proliferation In Human Osteosarcoma

Objective:The first objective of this research is making comprehension to theexpression pattern of ZFX gene in tissue samples and its clinicalcorrelation. In the second place, we have constructed a vector of shRNAlentivirus targeting ZFX gene and applied to osteosarcoma cell lines whichare Saos-2and MG63in order to reveal the functional role of ZFX in vitro.Lastly, the effects of ZFX silence on the cell proliferation, colonyformatting ability and cell cycle was investigated, so that its function hasbeen revealed according to these works.Methods:The mRNA and protein expression of ZFX in20osteosarcoma and9normal tissue samples was detected via immunohistochemistry (IHC).Based on this assay, we explored the clinical correlation between theexpression of ZFX and the osteosarcoma occurrence.Reported sequence of shRNA targeting on ZFX gene were synthetizedand cloned into lentiviral vector. The recombinant lentivirus was packagedand infected the osteosarcoma cells. Real-time PCR and Western blot wereperformed to determine the expression level of ZFX at both mRNA andprotein levels.Saos-2and MG63cells infectedwith recombinant shRNA-expressinglentiviral particles were used to perform MTT growth curve and colony formation to evaluate the cell proliferation. As well as cell cycle changeswere investigated under the ZFX gene inhibition. Collectively, theseworks made the function of ZFX in osteosarcoma clear.Results:The IHC and statistical analysis results showed that higher expressionof ZFX gene in osteosarcoma tissues than in normal tissues Data from thequantitative PCR demonstrated that mRNA expression of ZFX in tumortissues were significantly higher than that in adjacenttissues.The recombinant shRNA lentiviral vectors targeting on ZFX weresuccessfully constructed and produced for virus particles. The ZFX mRNAand protein expression were significantly inhibited in human osteosarcomacell lines when infected by ZFX shRNA-expressing lentivirus.It was showed that the cell proliferation and colony formation abilityof Saos-2and MG63cells were inhibited. Cell cycle was significantlyblocked at G0/G1phase following the flow cytometric analysis. Thepopulation of cells at sub-G1phase was significantly induced followingthe knockdown of ZFX in osteosarcoma cells.Conclusions:In osteosarcoma and normal tissues, the specific expression of ZFXseems to be relevant to tumorigenesis. This being the case, ZFX may be amolecular symbol of osteosarcoma formation. Then, ZFX gene expressionin osteosarcoma cells was effectively down-regulated by lentivirus- mediated shRNA. Silence of ZFX inhibited the cell proliferation,regulated the cell cycle and even induced apoptosis in vitro. For such aconsideration, ZFX may be expected to be a new molecule target forcancer diagnostics and therapy.