OBJECTIVETo interfere in the Tca8113-CDDP cell line with siRNA of cyclin D1 and to investigate time and dose dependent gene silencing effect of siRNA of cyclin D1.

METHODSsiRNA of cyclin D1 was transfected into Tca8113-CDDP cells Fluorescent CY3 dye labeled siRNA GAPDH was used as the control. The transient transfecting efficiency was examined at 4, 24, 48 and 72 h. The relative quantity of the target RNA of cyclin D1 was analyzed with SYBR Green fluorescent dye kit by the Real-time PCR assay. The protein level of cyclin D1 was examined with Western blot. The changes of cisplatin sensitivity after treatment with siRNA cyclin D1 were examined with methyl thiazolyl tetrazolium (MTT) assay.

RESULTSThe optimized transfecting efficiency with CY3 labeled siRNA GAPDH in Tca8113-CDDP cells was over 90%. The silencing rate of cyclin D1 siRNA was 81.6% at 24 h, 80.7% at 48 h and 94.3% at 72 h. Dose-dependent manner of gene silencing effect was observed when the siRNA concentration was lower than 100 nmol/L, however, gene silencing effect reached its platform when the concentration was higher than 100 nmol/L. The protein levels of cyclin D1 at 24, 48 and 72 h after transfection decreased significantly, and so did the growth of cells. Inhibition rates of cell growth induced by cisplatin after administration with or without cyclin D1 siRNA were 58.4% and 34.8%, respectively.

CONCLUSIONSChemical synthesized cyclin D1 siRNA effectively silenced the expression of cyclin D1 gene in Tca8113-CDDP cells in vitro, with a time- and dose-dependent manner and target gene silence in cells increased its sensitivity to cisplatin.