[Effect of Na(+)/K(+)-ATPase alpha1 siRNA and ouabain upon cell cycle in human hepatoma HepG2 cell and its mechanism].
OBJECTIVE
To investigate the in vitro anti-cancer effects of ouabain combined Na(+)/K(+)-ATPase alpha1 siRNA upon HepG2 cells and its molecular mechanism.


METHODS
The Na(+)/K(+)-ATPase alpha1 subunit expressions of human hepatoma tissue, normal liver tissue and human hepatoma cell lines (HepG2, SMC7721 and Bel7402) were determined. The cells received different treatments (0.03 micromol/L siRNA, 0.1 micromol/L ouabain and combination). The proliferation of HepG2 was observed by CCK-8 assay. The activity of Na(+)/K(+)-ATPase was measured. The HepG2 cell cycle distribution was detected by flow cytometry. The mRNA and protein levels of Na(+)/K(+)-ATPase alpha1 subunit, MAPK1, Cyclin A, CDK2, PCNA and P21WAF1 were detected by quantitative RT-PCR and Western blot.


RESULTS
The gray value Na(+)/K(+)-ATPase alpha1 subunit in hepatoma tissue was 174.74 +/- 16.77 and 65.31 +/- 7.88 respectively in normal liver tissue. The Na(+)/K(+)-ATPase alpha1 subunit expression of hepatoma tissue was significantly higher than normal liver tissue(P < 0.05).The Na(+)/K(+)-ATPase alpha1 subunit expression level of HepG2 was higher than that in SMMC-7721 and Bel-7402 cells. The CCK-8 experiments demonstrated that siRNA, ouabain and combination could inhibit the HepG2 proliferation, and the combination group was different from the ouabain or siRNA group (P < 0.05). The 24, 48 and 72 h inhibitory rates of 0.03 micromol/L siRNA, 0.1 micromol/L ouabain and combination group were (17.4%, 20.3%, 24.3%), (37.5%, 44.3%, 51.2%) and (52.3%, 70.2%, 88.2%) respectively. The activity of Na(+)/K(+)-ATPase decreased in siRNA, ouabain and combination group. The S phase proportion of ouabain and combination group increased from 24.2% to 66.5% and 75.2% respectively. The 0.1 micromol/L ouabain and combination group could down-regulate the expression of Na(+)/K(+)-ATPsae alpha1, MAPK1, Cyclin A, CDK2, PCNA and up-regulate the expression of P21WAF1 in HepG2 cell.


CONCLUSIONS
The Na(+)/K(+)-ATPase alpha1 siRNA combined ouabain inhibits the proliferation of HepG2 cells by decreasing the expression of MAPK1 and induces the cell cycle S arrest by decreasing the production of Cyclin A/CDK2/PCNA complex and increasing the expression of P21(WAF1).
Xu
Zhong-wei
ZW
Medical College of Chinese People's Armed Police Forces, Key Laboratory for Biomarkers of Occupational and Environmental Hazard, Tianjin 300162, China.
Wang
Feng-mei
FM
Xu
Rui-cheng
RC
Hu
Wen-liang
WL
Chen
Xiao-yi
XY
D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D002453:Cell Cycle; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*; D010042:Ouabain / Q000494:pharmacology; D034741:RNA, Small Interfering; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*
D034741:RNA, Small Interfering; D010042:Ouabain; C499076:ATP1A1 protein, human; D000254:Sodium-Potassium-Exchanging ATPase