Objective     To investigate the influence of siRNA-mediated p15 silencing on cisplatin (CDDP) resistance in drug-resistant human hepatoma cell line HepG2/CDDP/2.0.      Methods     Drug-resistant human hepatoma cell line HepG2/CDDP/1.6 was obtained through culturing HepG2 cells in a series of RPMI1640 culture media having gradually increased concentrations of CDDP

with final CDDP concentration of 1.6 mg/L. MTT assay was used to test the sensitivity of HepG2/CDDP/1.6 c

ells to CDDP and IC

50

of CDDP was then determined. Drug-resistant cell line HepG2/CDDP/2.0 was obtained through culturing HepG2/CDDP/1.6 cells in the presence of 2.0 mg/L CDDP. Three siRNAs (Y1

Y2

and Y3) targeting p15 and a negative control (siRNA-F) were designed and synthesized. Lipofectamine

TM

2000 was used to transfect HepG2/CDDP/2.0 cells with siRNA-F at 30 nmol/L

60 nmol/L

and 90 nmol/L

and then laser-scanning confocal microscopy (LSCM) was used to find out the optimal transfection concentration of siRNA. HepG2/CDDP/2.0 cells were transfected with Y1

Y2

and Y3 at the optimal transfection concentration

and then RT-PCR was used to screen out the most effective siRNA. Flow cytometry was used to analyze the cell cycle distribution of HepG2/CDDP/2.0 cells. RT-PCR and Western blotting were used to analyze the expression of p15 mRNA and the expression of p15 protein and P-glycoprotein (P-gp) in transfected HepG2/CDDP/2.0 cells.      Results     HepG2/CDDP/1.6 cell line had a resistance index (RI) of 6.8 to CDDP

showing moderate drug resistance. The optimal transfection concentration of siRNA was 90 nmol/L. Among siRNAs Y1

Y2

and Y3 that all dramatically reduced p15 mRNA expression

Y3 exhibited the best interfering effect. At 24 h

48 h

and 72 h after transfection

the ratios of transfected HepG2/CDDP/2.0 cells in G

1

phase were 53.8%

51.6%

and 48.8%

respectively (P0.05). The mRNA and protein expression of p15 in transfected HepG2/CDDP/2.0 cells was significantly inhibited (P0.05)

while the expression of P-gp was significantly increased (P0.05).      Conclusion     In the drug resistance process of human hepatoma cells

the ratio of human hepatoma cells in G

1

phase increases over time. In drug-resistant HepG2/CDDP/2.0 cells

knockdown of p15 by RNA interference can decrease the ratio of cells in G

1

phase and increase their drug resistance by up-regulating P-gp expression.