Study On The Effects And Mechanism Of DCX And SPARC Co-expression On Radiosensitivity Of U-87MG Glioma Cell

Objective: In this study,we constructed a recombinant adenovirus co-expressing Doublecortin and Osteonectin（called Ad-DCX-SPARC）, then the effects ofAd-DCX-SPARC on cell growth, invasion and radiosensitivity of U-87MG cellswere examined and the potential mechanism was investigated.Methods: Recombinant adenovirus Ad-DCX-SPARC was constructed andamplified. The function of adenovirus was confirmed by laser confocal microscope andflow cytometry (FCM). The expression levels of DCX and SPARC were measured byRT-PCR and Western blotting. We detect the effect of Ad-DCX-SPARC and withX-rays on the growth of glioma U-87MG cells by MTT assay. The invasive ability ofU-87MG transfected with Ad-DCX-SPARC were observed by Transwell assay. Colonyformation assay was used to determine the alternation of colony formation rate. FCMwas used to detect the apoptosis rate and the cell cycle distribution. The expression ofapoptosis-related protein Bad, Bax, Bcl-2and invasion-related protein MMP-2,MMP-9was determined by Western blotting.Results: The infection dose of U-87MG cells used in this study was50MOI.DCX and SPARC were transcribed and expressed successfully in U-87MG cells. Thegrowth of U-87MG glioma cells was significantly inhibited by Ad-DCX-SPARC,especially treated together with X-ray irradiation. The mean lethal dose, quasithreshoulddose and2Gy surviving fraction in the transfected cells were lower than the controlgroup. Ad-DCX-SPARC has no effect on cells without irradiation,but could augment theapoptosis induced by X-ray irradiation. Cell cycle analysis showed Ad-DCX-SPARCcould lead to G2phase accumulation, but downregulate the enhanced G2phaseaccumulation after irradiation. Transwell assay revealed the number of cells through themembrane of Ad-DCX-SPARC group was less than Ad-GFP group. Bad, Bax and Bcl-2protein expression of Ad-DCX-SPARC group were not changed significantly,but with irradiation of8Gy, Bad and Bax protein levels increased and Bcl-2protein decreased.Conclusion: The adenovirus co-expressing DCX and SPARC was successfullyconstructed and can transfected U-87MG cells efficiently. Ad-DCX-SPARC couldinhibit the growth and cell invasion and diminish the radiosensitivity of human glioma.Ad-DCX-SPARC could augment the apoptosis and reduce G2accumulation induced byX ray irradiation. upregulate Bad, Bax and downregulate Bcl-2may account for theincreased radiosensitivity. Downregulation of MMP-2, MMP-9may be one of themechanisms of inhibition of U-87MG cells invasive.