[Construction of miR-508-5P lentiviral vector and its targeted effect on S-phase kinase-associated protein-2 gene].

ABSTRACT

OBJECTIVE: To construct a lentiviral vector overexpressing miR-508-5p and verify its targeted regulating effect on S-phase kinase-associated protein-2 (SKP2) gene. METHODS: The stem-loop structure RNA of miR-508-5p was obtained by chemical synthesis and cloned into the linearized pSicoR plasmid. The positive recombinants, which had been identified by restriction enzyme digestion and DNA sequencing method, were transfected into HEK293T cells. Meanwhile, the potential target gene of SKP2-3'UTR, which was complementary with miR-508-5p, was obtained and cloned into the linearized pMIR-Report plasmid. The targeted effect of miR-508-5p on SKP2 gene was verified by the relative luciferase activity measurement, Western blotting and real-time PCR. RESULTS: The PCR, restriction enzyme digestion and DNA sequencing analysis demonstrated that the recombinant plasmids of pSicoR-miR-508-5p and pMIR-Report-SKP2 3'-UTR were constructed successfully. And it was showed that over-expression of miR-508-5p suppressed the mRNA and protein expression level of SKP2 significantly (P<0.05); and suppression of miR-508-5p expression increased the mRNA and protein expression level of SKP2 significantly (P<0.05). CONCLUSION: We have successfully constructed the lentiviral vector containing miR-508-5p gene. MiR-508-5p can suppress SKP2 gene expression by targeting the specific sequence of SKP2-3'UTR.