Construction and expression of siRNA against urokinase type plaminogen activator (uPA)

    HUANG Hongª²Yan1, SUN Qiang2, LIU Jiaª²Yun1, LI Qingª²Xia1, BAO Wei1, JIA Linª²Tao1,WANG Chenª²Ji1,YANG Anª²Gang1

    1Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fourth Military Medical University, Xi¬ðan 710033, China, 2Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing 100071, China

    ¡¾Abstract¡¿ AIM: To construct vectors expressing siRNA against urokinase type plaminogen activator (uPA) and to express them in mammalian cells. METHODS: Oligos of 64 base pairs for hairpin RNA targeting uPA were chemically synthesized and annealed. The siRNA constructs for uPA, produced by inserting the annealed oligos into the downstream of H1 promoter of linearized pSUPER, were transfected into MDAª²MB 231, a breast cancer cell line expressing high level uPA. After the siRNA constructs were confirmed by restriction digestion and DNA sequencing, RTª²PCR was performed to monitor the expression and interference efficiency of siRNAs against uPA. RESULTS: Restriction digestion and DNA sequencing showed that the siRNA constructs for uPA were successfully produced and the expressed siRNA could effectively downª²regulate the expression of uPA. CONCLUSION: Vectors expressing hairpin RNA against uPA are successfully produced, which will facilitate further studies of uPA function and its application in the treatment of tumors. ......