Construction of recombinant lentiviral vector of shRNA targeting SIRT1 gene and its effects on the proliferation of breast cancer cell

Abstract:

Objective：To construct the lentiviral vector expressing shRNA targeting silent information regulator 1（SIRT1） gene and to investigate its effects on proliferation and apoptosis of breast cancer cell MCF-7. Methods：Two shRNA sequences targeting SIRT1 gene were designed and synthesized and were connected to pLentilox3.7-green fluorescent protein（GFP） vector to reconstruct lentivi－ral plasmids pshSIRT1-1 and pshSIRT1-2.Meanwhile，shRNA negative control pshCont targeting no gene was constructed. Recombi－nant lentiviral vector expressing shRNA together with pLP1，pLP2 and pLP/VSVG were co-tranfected into 293FT cells and lentivirus was produced. The titer of virus was tested according to the expression level of GFP in 293FT cells． MCF-7 cells were infected with recombinant lentivirus and the silence efficiency was measured by real-time PCR and Western blot. Proliferation and apoptosis of MCF-7 cells were determined by trypan blue exclusive assay and flow cytometry respectively. Results：Lentiviral vectors containing shRNAs targeting SIRT1 gene were successfully established and corresponding lentiviruses were acquired. Real-time PCR and West－ern blot analysis showed that these two shRNA targeting SIRT1 gene significantly decreased SIRT1 mRNA（P=0.000 2） and protein levels. SIRT1 silencing resulted in marked decrease of proliferation and increase of apoptosis in MCF-7 cells（P=0.006 0）. Conclu－sions：Lentivirus-mediated RNA interference of SIRT1 can significantly inhibit MCF-7 cell proliferation and induce its apoptosis.