The Effect Of LEF1on The Synergy Of Emethylation Drugs And Paclitaxel Gainst Renal Cell Carcinoma

ObjectiveTo investigate the synergistic mechanism between5-Aza-2’-Deoxycytidine（DAC）andPaclitaxel (PTX) on RCC cell lines and to discuss the function of LEF1in this prosess.Methods1. Using MTT assay to determine the growth supression rate of RCC cells after72hourstreatment, and validate the cell growth inhibiting effects on the synergy of DAC and PTXagainst human renal clear cell carcinoma cell lines like ACHN, Caki-1, NC65, and usingradiation curve to validate the synergy effect in DAC and PTX against RCC cells.2. Synergy-associated gene LEF1has been is found out by gene expression microarray andwe will validate the mRNA expression of LEF1to discuss the function of LEF1on thesynergy effect in DAC and PTX by real time PCR. ACHN, NC65cell line is continuoustreatmented for72hours with DAC:0.5,1,2,4,8uM; PTX:1,2,4,8,16nM. Total RNAwas isolated using Rneasy mini kit (Qiagen), the First strand cDNA synthesis kit(Amasham Pharmacia) was used for reverse transcription, quantitative real-time PCR wasperformed using SYBR Green PCR Master Mix (Applied Biosystems) and analysefluorescence ration by IQ5.Results1. DAC and PTX have growth inhibiting effects against RCC cells.DAC and PTX all have growth inhibiting effects on three type of renal carcinoma celllines, and the cell surcival rate is decreased following the increasing of the drugconcentration and have dose dependent.2. The synergy of5-Aza-2’-Deoxycytidine（DAC）and PTX.The combination of DAC and PTX significantly supressed the growth of RCCsignificantly coppared to DAC or PTX alone. And using radiation curve to confirme thesynergy of the two agents can supress RCC cells proliferation significantly.3. LEF1is a associated gene in the synergy of DAC and PTX.Using qulitative PCR to determine the mRNA expression of LEF1was decreased in theprocessing of synergy of and decreased significantly compared to DAC or PTX alone,LEF1is an important downstream gene of the synergy of DAC and PTX against RCC. Conclusion1. DAC can increase the sensibility of the RCC cells to PTX and have the synergy potency.2. We have found out LEF1will be a important target gene in the synergy of DAC and PTXagainst RCC by gene expression microarray, in this study we validate this conclusion withqulitative PCR, and discuss the feasibility of the synergy therapy in the future, thusprovide the important theory for the individuation therapeutic regimen.