ABSTRACT
Objective To construct and authenticate the lentiviral-mediated overexpression of mouse mitochondrial-targeted-8-oxoguanine DNA-glycosylase 1 (mito-OGG1) gene and the lentiviral-mediated short hairpin RNA (shRNA) down-regulation of OGG1 gene expression model in 661W cells.Methods Constructed the target plasmids,including pLenti-EF1a-EGFP-P2A-Puro-CMV-Mito-OGG1-3Flag (pLenti-OGG1-GFP) and pLKD-CMV-G&PR-U6-shRNA (pLKD-shRNA).293T cells were used to obtain green fluorescent protein (GFP)-tagged lentiviral vector of interest by using a second generation lentivirus packaging system.293T cells were also used for the virus titer estimation.The multiplicity of infection (MOI) of 661W cells was detected by fluorescence microscopy.A stable transfected cell line was screened by puromycin.Immunofluorescence was used to detect transfection efficiency and cytochrome C oxidase Ⅳ (COXⅣ)-OGG1 co-localization.OGG1 mRNA and protein expression levels were detected by real-time qantitative PCR (QPCR) and Western blot.Results Sequencing results showed that the inserted sequence in the over-expression plasmid was consistent with the mouse OGG1 (NM_010957.4) gene sequence in the gene library.The original lentiviral titer after packaging and purification was between 2.0× 107to 6.0× 107 TU/ml.The optimal MOI of 661W cells was 40,and puromycin with a concentration of 4.0 μg/ml successfully screened stable transformation.The transfection efficiency was up to 100％ after screening.Immunofluorescence demonstrated successful co-localization of OGG1 and COXⅣ.The relative expression levels of OGG1 mRNA in the blank control group,OGG1 group,overexpression control group,shRNA group and low expression control group were 1.000±0.000,41.581±12.206,0.888±0.056,0.239±0.121 and 1.081±0.083,and the relative expression levels of OGG1 protein were 1.029±0.153,1.657 ± 0.237,0.752 ± 0.143,0.471 ± 0.149 and 1.036 ± 0.185,respectively,with significant differences between them (F=44.654,30.948;both at P＜0.05),the relative expression levels of OGG1 mRNA and protein in the OGG1 group were significantly higher than those in the overexpression control group,the relative expression levels of OGG1 mRNA and protein in the shRNA group were significantly lower than those in the lower expression control group,with significant differences between them (all at P＜0.05).Conclusions The mitoOGG1 overexpression and OGG1 knockdown models of 661W cells are successfully constructed,which provides the preliminary experimental basis for follow-up study.
ABSTRACT
Objective To estimate the relationship of the functional single nucleotide polymorphism (SNP) rs1052133 in the 8-oxoguanine DNA glycosylase 1 (OGG1) gene with vitiligo in a Chinese Han population.Methods Blood samples were collected from 800 patients with vitiligo and 800 healthy human controls,and subjected to genomic DNA extraction.PCR-restriction fragment length polymorphism (PCR-RFLP) analysis was performed to analyze the genotype of the SNP rs1052133 in the OGG1 gene.The relationship between the SNP and the risk of vitiligo was evaluated by chi-square test and unconditional logistic regression analysis.Enzyme linked immunosorbent assay (ELISA) was carried out to assess the serum level of 8-hydroxydeoxyguanosine (8-OHdG) in 83 patients with vitiligo and 83 healthy human controls,then,t test was used to compare the serum 8-OHdG level between the patients and controls.Results The frequency of CC,CG and GG genotype of the SNP rs1052133 was 16.8％,54.0％ and 29.2％ respectively in the patients,21.4％,52.8％ and 25.8％respectively in the controls (x2 =6.26,P ＜ 0.05).Increased frequency of G allele of the SNP rs1052133 was observed in the patients with vitiligo compared with the controls (56.2％ vs.52.2％,x2 =5.16,P ＜ 0.05).A statistically increased risk of vitiligo was associated with the CG (x2 =3.98,P ＜ 0.05,adjusted odds ratio 1.31,95％ confidence interval:1.01-1.70) and GG (x2 =6.01,P ＜ 0.05,adjusted odds ratio 1.45,95％ confidence interval:1.08-1.94) genotype of SNP rs1052133 compared with the CC genotype,which was more evident among the patients with the following characteristics:female,nonsegmental vitiligo,active vitiligo,long clinical course (＞ 12 months),a family history of vitiligo,and no accompanied autoimmune diseases.In addition,the patients with the CG or GG genotype of SNP rs1052133 had a higher serum 8-OHdG level than those with the CC genotype ((838.23 ± 294.11) μg/L vs.(593.84 ± 190.14) μg/L,t =3.63,P ＜ 0.01).Conclusions The SNP rs1052133 in the OGG1 gene may be responsible for the development of vitiligo in Chinese Han populations,which is likely to be associated with defects in DNA repair.