Study On Effects Of DCX And SPARC Co-expression On Glioma Cell Growth、invasion And Radiosensitivity With Underlying Its Mechanisms

ObjectiveTo construct a recombinant adenoviral vector co-expressing Doublecortin(DCX)and Secreted protein acidic and rich in cysteine(SPARC) and study its enhancedanti-tumor and radiosensitivity effects.Methods1. DCX and SPARC were amplified by PCR using cDNA as template.pAdTrack-CMV-DCX-polyA+promoter-SPARC double genes co-expression transfervector was constructed and identified by PCR, double endonuclease digestion, andDNA sequencing;2. pAdTrack-CMV-DCX-polyA+promoter-SPARC transfer vector was linearizedwith PmeI digestion and further co-transfected with pAdEasy-1backbone vector intobacteria BJ5183competent cells for homologous recombination;3. The production of pAdEasy-1-pAdTrack-CMV-DCX-polyA+promoter-SPARC(pAd-DCX-polyA+promoter-SPARC) homologous recombinant plasmid purified fromthe above BJ5183cells was transfected into the bacterial DH5α cells to abundantlyamplify pAd-DCX-polyA+promoter-SPARC plasmids, then they were linearized withPacI digestion and transfected into the human embryonic kidney293(QBI-293A) cellsby Lipofectamine2000, leading to packaging of the recombinant adenovirusesAd-DCX-polyA+promoter-SPARC(Ad-DCX-SPARC).First generation virus-containingsupernatants was used for obtaining large quantities of recombinant adenoviral vectorsafter several cycles of transfection and amplifying; 4. The best MOIs of Ad-DCX-SPARC were chosen by Ad-DCX-SPARC infectingU251and A172glioma cells;5. A series experimental methods which tested cell proliferation、invasion andradiation sensitivity were carried out to study the effects of increased DCX and SPARCco-expression for glioma cell growth, invasion and radiosensitivity, and analyzed itspossible mechanism.Results1. The double promoters mediated DCX and SPARC gene co-expressionadenoviral vector Ad-DCX-SPARC was successfully constructed. It could infect gliomacells U251and A172with high efficiency at the best infection doses of20MOI and100MOI respectively;2. Adenovirus-mediated DCX and SPARC gene co-expressionsignificantly inhibited the growth of glioma cells, weakening the role of SPARCpromoting glioma invasion in vitro;3. DCX and SPARC double gene co-expressioncould remarkably inhibit cell colony formation of U251and A172glioma cells postirradiation;4. DCX and SPARC double gene co-expression showed little effect onapoptosis in glioma cells, but it could promote apoptosis significantly after combinationwith radiation;5. Increasement of DCX and SPARC expression could enhanceproportion of G2phase cells before radiation. When combined with X-ray radiation, itwould decrease G2phase cell cycle arrest after irradiation to reduce repairment of DNAdamage with a result of more death of cells ultimately;6. Molecular mechanismanalysis showed that Ad-DCX-SPARC and radiation combination could remarkablyup-regulated the expressions of bax and bad, and down-regulated the expression ofbcl-2.Conclusion:1. Our study successfully constructed DCX and SPARC gene co-expressionadenovirus vector Ad-DCX-SPARC, it could significantly inhibit the growth of gliomacells and weakening the role of SPARC promoting glioma invasion in vitro;2. Ad-DCX-SPARC combined with X-ray radiation inhibited glioma cell colony formation, increased G2phase cells arrest before irradiation and enhancedirradiation-induced apoptosis. In addition, it could also weaken the G2phase cell arrestafter glioma irradiation, so that DNA damage repair was decreased with more gliomacells death. All the above results increased cell radiosensitivity of glioma;3. Ad-DCX-SPARC and radiation combination induced glioma cell apoptosisthrough up-regulating bax、bad, down-regulating bcl-2and so on apoptosis relatedgenes.