Construction of shRNA expression vector targeting DJ-1 and screening of stably transfected HepG2 cells

Objective To construct the DJ-1 shRNA eukaryotic expression vectors and obtain the stably transfected HepG2 cell lines. Methods Targeting DJ-1 gene interference sequence was synthesized and inserted into pGenesil-1 vector. The recombinant RNA interference (RNAi) vectors were transfected into HepG2 cells with Lipofectamine 2000. The transfected cells were persistently screened under G418 (450 mg/L), and isolated with a limited dilution for 8 weeks. Green fluorescence of the stably transfected HepG2 cell lines were observed under the fluorescence microscopy. The mRNA and protein expression of DJ-1 in the selected clones was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Results Sequencing indicated that RNAi eukaryotic expression vectors targeting DJ-1 had correct reading frame and nucleotide sequence, and the positive rate of stable transfected HepG2 cells was more than 75% per clone. RT-PCR revealed that DJ-1 shRNA effectively silenced mRNA expression level of DJ-1. Western blotting discovered that DJ-1 shRNA obviously suppressed protein expression level of DJ-1 with the inhibition rate being 98.78% (P ＜0. 05). Conclusion DJ-1 shRNA eukaryotic expression vectors were successfully constructed, and the establishment of stably transfected HepG2 cell lines provided a original route for exploring the mechanism of DJ-1 in human hepatocellular carcinoma cells further. Key words: DJ-1;  RNA interference;  Carcinoma,hepatocellular