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BACKGROUND:Overexpression ofα-synuclein can induce cell apoptosis.RNA interference(RNAi) may block specific gene function and cause gene silencing. OBJECTIVE:To construct a specific and effective RNAi plasmid for theα-synuclein gene and investigate if RNAi can block apoptosis in HEK293 cells,induced by overexpression of wild-typeα-synuclein. DESIGN,TIME AND SETTING:A contrast experiment based on genetically engineered cytobiology was performed at the State Key Lab of Medical Genetics of China,Xiangya Medical College of Central South University,between October 2004 and October 2008. MATERIALS:HEK293 cells and pBSHH1 plasmid were provided by the State Key Lab of Medical Genetics of China;OligDNA sequence by Sagon Bioengineering Company,Shanghai; Lipofectamine 2000 by Invitrogen,USA;α-synuclein monoclonal antibody,Hoechst 33258,and MTT; by Sigma,USA;Horseradish peroxidase-coupled goat anti-rat IgG by KPL,USA;FACSan flow cytometry by BD,USA. METHODS:Four target sites were used to construct hairpin RNA pBSHH1 vectors-pSYNi-1, pSYNi-2,pSYNi-3 and pSYNi-4-which were cloned in the pBSHH1 plasmid.HEK293 cells were transfected using Lipofectamine 2000.In addition,a non-transfect group and a negative plasmid transfect group were established.The cultured HEK293 cells were processed as follows: transfection of blank plasmid(blank control group),transfection ofα-synuclein-pEGFP and RNAi negative vector(negative control group),and transfection ofα-synuclein-pEGFP and pSYNi-1 (transfection group).Cells in all groups were transfected with Lipofectamine 2000 for 48 hours. MAIN OUTCOME MEASURES:Expression ofα-synuclein mRNA and protein were detected by RT-PCR and Western blot.Cell morphology was observed under an inverted fluorescence microscope;cell viability was measured using MTT method;and cell apoptosis was determined with Annexin V-PE flow cytometry. RESULTS:α-synuclein mRNA and protein expressions were significantly decreased in the pSYNi-1 group when compared with the non-transfect and negative plasmid transfect groups(P<0.05).The expressions were partially decreased in the pSYNi-2 group,but there was no significant difference in the pSYNi-3 and pSYNi-4 groups.Hoechst staining indicated that cell nuclei were enlarged in the negative control group,coloring was not uniform,and chromatin was accumulated and appeared spot-like.The nucleus coloring was uniform in the transfection group compared to negative control group.Cell viability in the negative control group was significantly lower than blank control group with cell apoptosis being significantly increased(P<0.05).In comparison with negative control group, cell viability was significantly increased in the transfection group and cell apoptosis was significantly decreased(P<0.05). CONCLUSION:pSYNi-1 can inhibitα-synuclein gene expression and block apoptosis of HEK293 cells induced by overexpression of wild-typeα-synuclein.
BACKGROUND: Overexpression of α-synuclein can induce cell apoptosis. RNA interference (RNAi) may block specific gene function and cause gene silencing. OBJECTIVE: To construct a specific and effective RNAi plasmid for the α-synuclein gene and if if RNAi can block apoptosis in HEK293 cells, induced by overexpression of wild-type α-synuclein. DESIGN, TIME AND SETTING: A contrast experiment based on genetically engineered cytobiology was performed at the State Key Lab of Medical Genetics of China, Xiangya Medical College of Central South University, between October 2004 and October 2008. MATERIALS: HEK293 cells and pBSHH1 plasmid were provided by the State Key Lab of Medical Genetics of China; Olig DNA sequence by Sagon Bioengineering Company, Shanghai; Lipofectamine 2000 by Invitrogen, USA; α-synuclein monoclonal antibody, Hoechst 33258, and MTT; by Sigma, USA; Horseradish peroxidase-coupled goat anti-rat IgG by KPL, USA; FACSan flow cytometry by BD, USA. METHODS: Four target sites were used to const ruct hairpin RNA pBSHH1 vectors-pSYNi-1, pSYNi-2, pSYNi-3 and pSYNi-4-which were cloned in the pBSHH1 plasmid. HEK293 cells were transfected with Lipofectamine 2000. In addition, a non-transfect group and a negative plasmid transfect group were established the cultured HEK293 cells were processed as follows: transfection of blank plasmid (blank control group), transfection of a-synuclein-pEGFP and RNAi negative vector (negative control group), and transfection of a-synuclein- pEGFP and pSYNi- 1 (transfection group) .Cells in all groups were transfected with Lipofectamine 2000 for 48 hours. MAIN OUTCOME MEASURES: Expression of α-synuclein mRNA and protein were detected by RT-PCR and Western blot. Cell morphology was observed under an inverted fluorescence microscope; cell viability was measured using MTT method; and cell apoptosis was determined with Annexin V-PE flow cytometry. RESULTS: α-synuclein mRNA and protein expressions were significantly decreased in the pSYNi-1 group when compared with the non-tranThe expressions were partially decreased in the pSYNi-2 group, there was no significant difference in the pSYNi-3 and pSYNi-4 groups. Hoechst staining indicated that the cell nuclei were enlarged in the negative control group, coloring was not uniform, and chromatin was accumulated and appeared spot-like. The nucleus coloring was uniform in the transfection group compared to negative control group. Cell viability in the negative control group was significantly lower than blank control group with Cell apoptosis was significantly increased (P <0.05) .In comparison with negative control group, cell viability was significantly increased in the transfection group and cell apoptosis was significantly decreased (P <0.05). CONCLUSION: pSYNi-1 can inhibitα-synuclein gene expression and block apoptosis of HEK293 cells induced by overexpression of wild-type α-synuclein.