S phase kinase-associated protein 2 regulates rat mesangial cells proliferation / 中华肾脏病杂志

ABSTRACT

Objective To explore whether the change of S phase kinase-associated protein 2 (Skp2) expression could regulate mesangial cell proliferation. Methods Skp2 siRNA and control siRNA, pIRES-GFP-Skp2 plasmid and pIRES-GFP plasmid were designed and synthesized. Cell transfection was performed by Lipofectamine 2000. Skp2 mRNA and protein levels were detected by semiquantitative PCR and Western blotting respectively. Primary culture rat mesangial cells were divided into 6 groups 0%FCS, 20%FCS, 10%FCS+pIRES-GFP plasmid, 10%FCS+pIRES-GFP-Skp2 plasmid, 20%FCS+control siRNA, 20%FCS+Skp2 siRNA. Cell number was detected by MTT. S phase entry was measured by BrdU labeling. Cell cycle profile was determined by flow cytometric analysis. Results Skp2 mRNA level was significantly down-regulated by Skp2 siRNA compared to control siRNA. Skp2 protein level increased after pIRES-GFP-Skp2plasmid transfection compared to pIRES-GFP plasmid. MTT, BrdU labeling and cell cycle profile demonstrated that cell number (A 0.419±0.088 vs 0.305±0.036, P＜0.01) and S-phase cells (BrdU labeling positive cell 0.21±0.04 vs 0.15±0.03, P＜0.01;S-phase cell number20.18±0.64vs 14.33±0.37, P＜0.01) obviously increased after Skp2 plasmid transfection, while decreased after Skp2 siRNA transfection compared to control groups (A 0.328±0.069 vs 0.482±0.133, P＜0.01;BrdU labeling positive cell 0.17±0.01 vs 0.24±0.00, P＜0.01; S-phase cell number 16.52±0.75vs 23.81 ±1.25, P＜0.01). Conclusion Over-expression of Skp2 stimulates mesangial cell proliferation while down-regulated expression of Skp2 inhibits mesangial cell proliferation.