Studies On Inhibition Of Epidermal Growth Factor Receptor In Non-small-cell Lung Cancer By RNA Interference

Part Ⅰ:Downregulation of enhanced green fluorescence protein gene expression by RNA interference in mammalian cellsOBJECTIVE: RNA interference (RNAi) is a recently observed process by whichdouble-stranded RNA (dsRNA) directs sequence-specific degradation of messengerRNA (mRNA) in animal and plant cells. The aim of this study was to investigate theefficiency and mechanism of RNAi induced by chemically synthetic smallinterference RNA (siRNA) in mammalian cells.METHODS: We transduced 293T cells by enhanced green fluorescence protein(EGFP) gene via lentivirus and obtained EGFP positive cells i.e. 293T/GFP cells.Then we introduced chemically synthetic 21-nucleotide siRNA duplexes into293T/GFP cells by means of TransIT-TKO, Oligofectamine reagent, andLipofectamine 2000 respectively. The effects of gene silencing were observed by bothfluorescence microscopy and flowcytometry. To study whether the silencing effectwas processed in a dose and time-dependent manner, we transfected the 293T/GFPcells by means of Lipofectamine 2000 with 0.01, 0.02, 0.04, 0.08, 0.16 μmol/LdsRNA–EGFP for a 48 h expossure, or transfected the cells with 0.08 μmol/LdsRNA–EGFP at four preselected time points i.e. 12 h, 24 h, 48 h, 72 h.RESULTS: EGFP expression was significantly and specifically inhibited by thecorresponding dsRNA, but not by unrelated dsRNA. In three different vectors,Lipofectamine 2000 demonstrated the highest transfection efficiency with a 48 hexposure. The decrease in EGFP fluorescence intensity was approximate 80%.Although TransIT-TKO and Oligofectamine displayed similar trends, thetransfections were inefficient (41.02% for TransIT-TKO, 37.45% for Oligofectamine),and often toxic. The results also exhibited that siRNA inhibited the EGFP geneexpression in a dose and time-dependent manner.CONCLUSION: The Lipofectamine2000 was a better transfection reagent for RNAi.RNAi induced by chemically synthetic siRNA processed in a time and dosedependent manner, and hence, RNAi pathway seems operative in mammalian embryocells. RNAi may be developed into a potential tool for gene therapy. 4Keywords: RNA interference; small interference RNA; double stranded RNA;enhanced green fluorescence proteinPart Ⅱ: Effects of RNA interference on epidermal growth factor receptor expression in non-small-cell lung cancer cells in vitroOBJECTIVE: To investigate whether RNA interference (RNAi) originated by smallinterference RNA (siRNA) could induce gene silencing in non-small-cell lung cancer(NSCLC) cells as well as assess the degree of epidermal growth factor (EGF)receptor gene silencing and its effect on functional outcome.METHODS: To determine whether EGF receptor gene is overexpressed in NSCLCcells as well as in a wide range of other cancer cells. Then NSCLC cell lines A549,SPC-A1 were transfected with target sequence-specific dsRNA as well as variouscontrols. Immune fluorescent labeling, flowcytometry and Western Blot were used tomonitor the reduction in the production of the EGF receptor protein. Quantitativereverse-transcriptase PCR was used to detect the silencing of the EGF receptor genelevel. Cell count, colony assay, scratch assay, MTT assay, cell cycle analysis, andELISA were used to assess the function effects of RNAi.RESULTS: We have demonstrated here EGF receptor gene was overexpressed inNSCLC cells. In A549 cell line, we displayed sequence specific silencing of the EGFreceptor with 71.31% of down-regulation of EGF receptor protein production and37.04% of silencing of EGF receptor gene. The reduction in EGF receptor causedgrowth inhibition of the cell, i.e.: reducing the total cell numbers by 85.0%, andcolony number by 63.3%. This also retarded the migration of A549 by more than 80%both at 24h and at 48h, enhanced the chemosensitivity to cisplatin by four-fold. Cellcycle analysis showed that dsRNA-EGFR induced accumulation of cells in G0-G1phase by 12.67% with a decrease in...