Construction of recombinant lentiviral vector of shRNA targeting SIRT1 gene and its effects on the proliferation of breast cancer cell
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Construction of recombinant lentiviral vector of shRNA targeting SIRT1 gene and its effects on the proliferation of breast cancer cell
Journal of Chongqing Medical UniversityVol. 36, Issue 5, Pages: 474-477(2013)
作者机构:
重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆,400016
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Published:2013,
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TAO Ying, CHEN Ke, SONG Chunli, et al. Construction of recombinant lentiviral vector of shRNA targeting SIRT1 gene and its effects on the proliferation of breast cancer cell. [J]. Journal of Chongqing Medical University 36(5):474-477(2013)
DOI:
TAO Ying, CHEN Ke, SONG Chunli, et al. Construction of recombinant lentiviral vector of shRNA targeting SIRT1 gene and its effects on the proliferation of breast cancer cell. [J]. Journal of Chongqing Medical University 36(5):474-477(2013)DOI:
Construction of recombinant lentiviral vector of shRNA targeting SIRT1 gene and its effects on the proliferation of breast cancer cell
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.
关键词
慢病毒沉默信息调节因子1(SIRT1)乳腺癌细胞RNA干扰
Keywords
lentivirussilent information regulator 1(SIRT1)breast cancer cellRNA interference
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Related Institution
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