ABSTRACT
Objective:To investigate the effect of survivin-siRNA plasmid on survivin expression in human lung cancer cell line A549, and to observe its effect on the apoptosis, proliferation, and chemosensitivity of A549 cells. Methods: pSilencer-survivin-siRNA (survivin-siRNA) plasmid was constructed using pSilencer-U6 plasmid and was transfected into A549 cells. Expression of survivin mRNA and protein was examined by RT-PCR and Western blotting analysis, respec-tively. Apoptosis and proliferation of A549 cells were examined by DAPI staining and MTT, respectively. Results: Sur-vivin-siRNA plasmid was successfully constructed, and it significantly inhibited survivin mRNA and protein expression in A549 cells. Survivin-siRNA transfection induced apoptosis, inhibited proliferation and increased chemosensitivity of A549 cells to cisplatin. Conclusion: pSilencer-survivin-siRNA can silence survivin expression in A549 cells and subsequently inhibit proliferation, promote apoptosis, and enhance chemosensitivity of A549 cells to cisplatin. Survivin may serve as a potential target for gene therapy of lung cancer.
ABSTRACT
@#Objective To investigate the effects of RNA interference (RNAi) on Caspase-3 expression.Methods According to the gene sequence and the secondary structure characteristics of Caspase-3, three siRNA （Si-Caspase-3-1, Si-Caspase-3-2 and Si-Caspase-3-3） were designed with 21 basic group, and siRNAs against Caspase-3 gene were synthesized. With the liposome conduct, the synthesized siRNAs were transferred into neuroglia cells. After the cells crept the slices, cells were dyed with fluor Hoechst33258; the apoptotic cells were stained with TUNEL, the normal neuroglia cells were as control, the Caspase-3 protease activity were detected with hemi-quantitative RT-PCR and Western blot.Results The synthesized siRNAs could inhibit the expression of Caspase-3 gene. For Si-Caspase-3-3, the expression inhibition rate of Caspase-3 mRNA was 86.32% in neuroglia cell.Conclusion The synthesized siRNAs can inhibit the expression of Caspase-3 gene at cell level.
ABSTRACT
Objective To investigate the inhibitive effects of biosynthesized short hairpin RNA(shRNA)on the duplication of Influenza A virus(IAV)in human bronchial epithelium(HBE)cells.Methods NP-shRNA and PA-shRNA targeting to the highly conservative sequences of IAV nucleoprotein(NP)and acidic RNA polymerase(PA)were designed and biosynthesized by in vitro transcription,and then were inserted into the plasmid pGenSil-1.After sequencing analysis,the recombinant plasmids NP-shRNA and/or PA-shRNA were transduced into HBE cells followed by infected with IAV A/PR/8/34 H1N1(A/PR8)virus.The cytopathogenic effect(CPE)of the HBE cells,hemagglutination assay(HA)and 50% tissue culture infective dose(TCID50)titer of A/PR8 in the culture supernatants were determined respectively.The mRNA and protein expressions of NP and PA were detected by RT-PCR and Western blotting respectively,and the results were compared in order to evaluate the inhibitive efficiency of NP-shRNA and/or PA-shRNA to A/PR8 duplication in HBE cells.Results The content of NP-shRNA and PA-shRNA biosynthesized by in vitro transcription were respectively 59.4 nmol and 50.6 nmol in each 100 ?l.The purity was more than 2.0 and the sequences were verified to be correct after sequencing analysis.The CPE of HBE cells and the virus titer in the culture supernatants of cells transfected with NP-shRNA,PA-shRNA or NP-shRNA+PA-shRNA were markedly lower than those of the control groups.In 3 h after 1 TCID50 A/PR8 infection,the mRNA level of NP in NP-shRNA group,the mRNA level of PA in PA-shRNA group and those in NP-shRNA+PA-shRNA group were 41.7%,43.4%,68.5% and 73.7% respectively,lower than those of the control group.At 48 h after 1 TCID50 A/PR8 infection,NP synthesis were 92.3%,84.0% and 91.7% respectively of those of the control group.Conclusion Anti-IAV NP-shRNA and PA-shRNA are successfully prepared by in vitro transcription.Both of those markedly inhibit the reproduction of IAV A/PR8 and produce a favorable protective effect on HBE cells.