Abstract:

BACKGROUND: Rac1, which is expressed in a variety of tumors, regulates the invasion and proliferation of cancer cells through signal transduction.
OBJECTIVE: To construct a RNA interference lentiviral vector that is capable to knock down Rac1 gene, and to detect its interference efficiency.
METHODS: The gene expression of Rac1 in tongue cancer cells was detected by western blot. The Oligo DNA containing target sequence was synthesized according to the previously confirmed effective sequence of small interfering RNA targeting Rac1 gene. Double-stranded DNA was constructed after annealing, and was cloned into the pMagic4.1 vector after digest with AgeI/EcoRI to construct a lentiviral vector which express short hairpin RNA. Positive clones were identified using PCR. The plasmids and packaging plasmids were cotransfected into 293T cells. The small interfering RNA expression cassettes were generated by real-time PCR; the successfully constructed plasmids were packaged by lentivirus and then transfected into Tca8113 cell. The interference effect of Rac1 gene expression was assayed by fluorescence quantitative PCR and western blot to explore its biological activity.
RESULTS AND CONCLUSION: RAC1 interference vector was constructed successfully. Western blot results showed that the protein expression of Rac1 was decreased significantly; Real-time PCR results showed that the inhibition rate of pLVT447 was up to 70%. A lentivirus-mediated RNAi vector containing Rac1 gene was successfully constructed, which provides effective small interfering RNA target sequence for the application of RNA interference in the targeting gene therapy of tongue cancer using Rac1.