ABSTRACT
To explore the potential of the anti-sense nucleic acid of CyclinD1 in lung cancer therapy, the expression vector containing the anti-sense nucleic acid of CyclinD1 was constructed and named pcDNA3.1-CyclinD1. The A549 cells were transfected with pcDNA3.1-CyclinD1 vectors. After being screened by G418, the stable expression positive clones were obtained. MTT method and flow cytometry technique were used to detect cell proliferation and apoptosis, respectively. The results showed the transfected cells exhibited significantly increased apoptosis and inhibited cell growth, compared with negative control and empty vector groups. To investigate the mechanism for anti-sense nucleic acid of CyclinD1 inducing A549 cells apoptosis, the expression levels of retinoblastoma protein (pRb), adenovirus E2 factor-1 (E2F-1), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and MMP-9 were detected by Western blot, and the results showed the expressions of these proteins were all decreased significantly in anti-sense nucleic acid of CyclinD transfected group, compared with those in negative control and empty vector groups. In a word, anti-sense nucleic acid of CyclinD1 induces the apoptosis of lung adenocarcinoma cancer cells, and the depressions of pRb, E2F-1, VEGF, MMP-2 and MMP-9 expressions may be the possible mechanism.
Subject(s)
Humans , Adenocarcinoma , Pathology , Apoptosis , Cell Line, Tumor , Cyclin D1 , Genetics , DNA, Antisense , Pharmacology , Genetic Vectors , Lung Neoplasms , Pathology , Matrix Metalloproteinase 2 , Metabolism , Matrix Metalloproteinase 9 , Metabolism , Recombination, Genetic , Retinoblastoma Protein , Metabolism , Transfection , Vascular Endothelial Growth Factor A , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To evaluate the role of RNA interference (RNAi) in silencing the enhanced green fluorescent protein (eGFP) expression in 293T and Mel cells.</p><p><b>METHODS</b>Nested-PCR was used to amplify H1 promoter from human 293T cells for driving RNAi synthesis. RNAi vectors (TR1) for silencing the eGFP expression was constructed. The eGFP vector and RNAi vector (TR1) were then co-transfected into the 293T and Mel cells, in which the silencing effect on eGFP expression was investigated by fluorescence microscopy, reverse transcription-PCR(RT-PCR), fluorescence-assited cell sorting(FACS) analysis and real-time RT-PCR.</p><p><b>RESULTS</b>RNAi could effectively reduce more than 50 percent of eGFP expression in 293T cells as well as in Mel cells.</p><p><b>CONCLUSION</b>The RNAi vector constructed in this way paper can effectively inhibit eGFP expression in cells.</p>