ABSTRACT
Aim To construct eukaryotic expression vector of shRNA(small hairpin RNA)for human SREBP-1(sterol regulation element binding protein-1)gene and explore its effects on lipid droplet formation in human renal proximal tubular epithelial cell line(HKC)under the stimulation of high glucose.Methods Two eukaryotic expression vectors of shRNA were constructed for human SREBP-1 gene.The HKC cells were transfected with negative control plasmid(pGenesil-1-HK)and two recombinant vectors(pGenesil-1-SREBP1-1 and pGenesil-1-SREBP1-2)and then were cultured under the stimulation of high glucose for about 48 h.The expression of SREBP-1 mRNA and FAS mRNA was detected by RT-PCR and SREBP-1 protein expression was investigated by Western blot.Lipid droplets were detected by Oil Red O staining.Results DNA sequencing showed that the target segments were successfully cloned into pGenesil-1 vector respectively.RT-PCR indicated that two recombinant vectors could inhibit the expression of SREBP-1 mRNA and FAS mRNA in HKC cells under the stimulation of high glucose.Similarly,SREBP-1 protein was also inhibited by the transfection with recombinant vectors.Oil Red O staining found that silencing of SREBP-1 gene resulted in lipid droplets decrease.Conclusions The eukaryotic expression vector of shRNA for human SREBP-1 gene was successfully constructed,and the expression of SREBP-1 was inhibited effectively by the expressed siRNA in HKC cells that resulted in lipid droplets decrease through FAS mRNA transcription inhibition.