Transfection of hairpin small interfering RNA expression vector targeting rat nuclear factor (NF) (κB) inhibits rat cell proliferation induced by NF-κB signal pathway activation

Objective: The aim of this work was to construct one small interfering RNA (siRNA) eukaryotic expression vector targeting rat nuclear factor (NF)κB p65 and identify its inhibition effect on cell proliferation according to its down-regulation of NF-κB pathway.

Methods: The p65siRNA expression vector "pGenesil-1.2-p65siRNA" and negative control plasmid "HK" were transfected into the cultured rat cells. After transfection, cells were divided into 4 treatment groups: 1) control cells cultured in complete. Dulbecco modified Eagle medium; 2) lipopolysaccharide (LPS) (1 μg/mL); (3) LPS (1 μg/mL) + HK-transfected; 4) LPS (1 μg/mL) + p65siRNA (pGenesil-1.2-p65siRNA). Thereafter, the protein levels of NF-κB p65 in the cells were detected by Western blotting at 72 hours after LPS stimulation. Furthermore, to observe cell proliferation, the proliferative rate of the cell growth was evaluated by the methylthiazolyl tetrazolium assay (at 24, 48, and 72 hours). The cell cycle distribution at 72 hours was detected by flow cytometry.

Results: p65siRNA effectively down-regulated the protein level of p65 (P < .05). Meanwhile, the proliferation of cells transfected with p65siRNA expression vector was significantly inhibited (P < .05), the ratio of cells at G(0)/G(1) stage markedly increased, and the proportion of cells at S stage was significantly decreased among transfected compared with control cells (P < .05).

Conclusions: p65siRNA effectively suppressed NF-κB, expression, inhibiting rat cell proliferation induced by NF-κB signal pathway activation.