Influence of RNA interference-induced repression of p53 expression on senescence in human skin fibroblasts / 中华皮肤科杂志

ABSTRACT

Objective To establish a cell line with repressed expression of p53 by transfecting a plasmid construct expressing short hairpin RNA(shRNA)targeting p53 into human skin fibroblasts(HSFs),and to evaluate the effect of repression of p53 expression on the senescence in HSFs.Methods The eukaryotic expressing plasmid pGCsi-p53 containing shRNA targeting p53 gene was transfected into HSFs with lipofectamine.Subsequently,the cells were selected by G418,and resistant cell clones were chosen and expanded.Reverse transcription-PCR and real time fluorescence-based quanitative PCR were performed to determine the expression of p53 gene,and Western blot to detect the expression of p53 protein in HSFs.The senescence in HSFs was evaluated by SA β-gal staining,and cell proliferation by methyl thiazolyl tetrazolium(MTT)assay.Results A HSF clone with repressed expression of p53 was established successfully.The expressions of p53 mRNA and protein were downregulated in transfected HSFs compared with untransfected HSFs(0.09 ± 0.03 vs.0.32 ± 0.04,0.11 ± 0.04 vs.0.84 ± 0.05,both P ＜ 0.01).The percentage of senescent cells was 13.47％ ± 1.01％ in the transfected HSFs,significantly lower than that in untransfected HSFs(18.10％ ± 0.66％,P ＜ 0.05).As MTT assay showed,the proliferation was accelerated in transfected HSFs compared with untransfected HSFs(P ＜ 0.05).Conclusions The repression of p53 expression decelerates the senescence in HSFs,but promotes the proliferation of HSFs.