Construction and identification of siRNA expression vector targeting KDR

    MIAO Yingª²Ye, LIU Jiaª²Yun, YI Jing, SU Mingª²Quan, SHEN Jianª²Jun, HAO Xiaoª²Ke

    Department of Clinical Laboratory, Xijing Hospital, Fourth Military Medical University, Xi¬ðan 710033, China, Department of Clinical Laboratory, Chinese PLA 152 Hospital, Pingdingshang 46700, China

     ¡¾Abstract¡¿ AIM: To construct pSilencerTM3.1ª²H1 small inferfering RNA (siRNA) expression vector targeting human KDR gene and to observe its silencing effect in the PC3 prostate cancer cell line. METHODS: The designed oligos targeting KDR were cloned into the pSilencerTM3.1ª²H1 neo vector, which was lineated after BamH¢ñ and Hind¢ó digestion. The recombimant vectors were confirmed by enzyme digestion analysis and DNA sequencing.The recombimant vectors of pSilencer 3.1ª²KDR1, pSilencer 3.1ª²KDR2 and pSilencer 3.1ª²KDR3 were transfected by liposomeª²mediated transfection into the PC3 prostate cancer cell line with high metastasis potential. At 72 h after transfection, the expression of KDR at the levels of mRNA and protein was detected by RTª²PCR and Western blotting. RESULTS: The pSilencerTM3.1ª²H1 siRNA expression vectors were constructed and confirmed after the enzyme digestion analysis and the DNA sequencing. The siRNA expression vectors of pSilencer 3.1ª²KDR1, pSilencer 3.1ª²KDR2 and pSilencer 3.1ª²KDR3,were successfully constructed.pSilencer 3.1ª²KDR3 was most effective in the 3 which downª²regulated 55% mRNA and protein of KDR at 72 h after transfection. CONCLUSION: pSilencerTM3.1ª²H1 siRNA expression vectors targeting KDR were successfully constructed. The expression of KDR gene was inhibited effectively in PC3 cells transfacted by pSilencer 3.1ª²KDR3,which laid a basis for its application in the treatment of prostate cancer. ......