The Influence Of The ZFX Gene Silence On The Ability Of Invasion And Migration Of Osteosarcoma Cells

Objective:The first objective of this research is making comprehension to the expressionpattern of ZFX gene in osteosarcoma (OS) tissue samples and its clinical value. In thesecond place, we have constructed a vector of siRNA lentivirus targeting ZFX gene andapplied to osteosarcoma cells of Saos-2and MG63，in order to verify the expression ofZFX in the transfected osteosarcoma cells. Lastly, the effects of ZFX down-regulationon the cell invasion and migration were investigated. Then, according to these works,the function of ZFX in the metastasis of osteosarcoma has been revealed.Methods:The mRNA and protein expression of ZFX in the osteosarcoma cell lines U2-OS,Saos-2, MG-63and SW1353and47cases of osteosarcoma tissue samples wereinvestigated by Western blot. The human brain glioma cell U251was used as thepositive control in Western blot investigation. Based on these assays, we explored theclinical correlation between the expression of ZFX and the characteristics ofosteosarcoma.Reported sequence of shRNAs targeting on ZFX gene were synthetized and clonedinto lentiviral vector ZFX-siRNA. The recombinant lentivirus was packaged andinfected the osteosarcoma cells of Saos-2、MG-63. Real-time PCR was performed todetermine the expression level of ZFX in Saos-2and MG-63after infection.The transwell test was performed to observe the invasion and migration ability ofSaos-2and MG-63cells infected by lentiviral vector siRNA-ZFX、Con and nonsensesequence Lv-shCon as the negative control respectively. Bacteria count was analyzedwith25uL flow cytometry and the movement experiment was analyzed with cellmovement kit. Results:The expression of ZFX gene can be seen both in osteosarcoma cell lines and inosteosarcoma tissue samples. Higher expression of ZFX gene was showed in MG63,Saos2and SW1353cell lines than in U2OS cell lines. ZFX was detected highexpression in51.1%(24/47) of patients with osteosarcoma. In addition, ZFX highexpression in osteosarcoma tissue significantly relates to the metastasis (P<0.05).The successful synthesized lentiviral vector identified by DNA sequence infectedosteosarcoma cell lines Saos-2、 MG-63.The plasmid carried GFP gene.Thesuccessfully transfected cells could be seen green fluorescence under the fluorescencemicroscope. The expression of GFP showed in more than90%cells under themicroscope demonstrated successful transfection.The effect of ZFX gene silence was verified by Real-time PCR.The result revealedthat the mRNA expression level of ZFX gene in siRNA-ZFX group was significantlylower than in CON and Lv-shCon groups.(P<0.05),while there was no statisticalsignificance between CON and Lv-shCON (P>0.05),which confirmed the shRNAlentivirus had significant effect of silence on ZFX gene.The result of transwell invasion assay indicated that the invasion rate ofsiRNA-ZFX group was notably lower than the Lv-shCon group (P<0.05),while therewas no statistical significance between CON and Lv-shCON (P>0.05).Conclusions:In osteosarcoma, the high expression of ZFX seems to be relevant to metastasis.The silence of ZFX gene significantly decreased the invasion and migration ability ofosteosarcoma cells of Saos-2、MG-63. ZFX gene is a potential target for the effectiveRNAi treatment of osteosarcoma patients.