Construction Of DAB2IP Expression Vector And Its Influence On Prostate Cancer LNCaP Cell DNA Synthesis And Apoptosis

ObjectiveTo construct the recombinant plasmid pEGFP-N1-DAB2IP and observe itseffect on DNA synthesis and apoptosis in human prostate cancer cell LNCaP.MethodsAccording to the gene sequence of DAB2IP, we designed a Pair ofPrimer.Total RNA was extracted from prostate cancer LNCaP cell.The full lengthof DAB2IP cDNA was amplified by PCR. After digestion by EcoRⅠandSalⅠ,ligation with pEGFP by T4DNA ligase, and transformation into DH5α,cultivation of bacteria and extract plasmid, the recombinant plasmid pEGFP-N1-DAB2IP was identified by enzyme digestion and DNA sequencing(). LNCaP cellswere transfected with pEGFP-N1-DAB2IP and the control plasmid pEGF-N1withliposome. Transfection result was observed by fluorescence microscopy. After48hours,the protein expression of DAB2IP was examined by Western blot. Brdu/PIincorporation was used to determine DNA synthesis of LNCaP cells. Annexin V-FITC/PI double staining were used to detect apoptosis.ResultsThe recombinant plasmid pEGFP-N1-DAB2IP was successfully appraisal byelectrophoresis and DNA sequencing, and successful transfection LNCaP cells.Western blot showed after transfection pEGFP-N1-DAB2IP group compareto pEGFP-N1group， the expression of DAB2IP protein level increasedsignificantly.BrdU/PI method showed pEGFP-N1group contrast pEGFP-N1-DAB2IPgroup BrdU＋cells have statistically significant (P <0.05), suggestingDAB2IP inhibit DNA synthesis of the LNCaP cells. After transfection pEGFP-N1group was compared with pEGFP-N1-DAB2IP group apoptotic cells difference was statistically significant (P <0.05),suggesting DAB2IP can induce the apoptosis of prostate cancer cells LNCaP.Conclusions1、 The experiment successful used RT-PCR technology to clon DAB2IPgene.2、 The recombinant plasmid pEGFP-N1-DAB2IP was successfullyconstructed and transfected LNCaP cells.3、DAB2IP gene overexpression could inhibit LNCaP cells DNA replicationto regulate cell proliferation and induce of cell apoptosis...