Reversion Of Expression Of The MDR1 And MRP In Human Tongue Carcinoma Cell Radiation-resistance Line By Antisense Oligonucleotides

Objective: ⑴To establishi a radiation-resistance human tongue carcinoma cellline ⑵To investigate the mechanism and effectiveness of phosphrothioateantisense oligonucleotides(ANOS) on suppressing Multidrug Resistance Gene1(MDR1) and multidrug resistance-associated protein gene(MRP) in radiation-resistance human tongue carcinoma cell line. ⑶To analyze the toxicity of ANOSand Lipofectamine to human tongue carcinoma cell line.Methods: ⑴Human tongue carcinoma cell radiation-resistance line(Tca8113) wasestablished with total dosage 20Gy. The expression of mutidrug resistance-associated protein P190 and P-gp was detected by immunohistochemical staining.⑵Cell line was transfected with synthetic ANOS complementing to the codingregion of MRP mRNA and MDR1 mRNA with Lipofectamine acting as carrier.The sense of NOS tranfection cell and no transfection cell were used as controls⑶The toxicity of ANOS and Lipofectamine was measured by MTT assay. ⑷The expression of MRP mRNA and MDR1 mRNA was detected by reversetranscription-polymerase chain reaction(RT-PCR) and the expression of P190 andP-gp was detected by flow cytometry(FCM).Results: ⑴Comparing with sense NOS transfection groups and no transfectiongroups, the expression of P190 and P-gp were declined in the ANOS tranfectiongroups, the level of expression of P190 and P-gp were relate to the concentration ofANOS and the time of transfection. The expression of P190 and P-gp in 0.5μmol/LANOS group were reduced more than other ANOS concentration groups(P<0.05),and the expression of P190 and P-gp on 24 hours ANOS group were the lowestamong those time groups(P<0.05).⑵Compared with sense NOS transfectiongroup and no transfection group, the expression of MRP mRNA and MDR1 mRNAin ANOS transfection groups were reduced after the radiation –resistant humantongue carcinomacell line was transfected , the level of expression of MRP mRNAand MDR1 mRNA were relate to the concentration of ANOS and the time oftransfection.The level of expression of MRP mRNA and MDR1 mRNA in 0.5μmol/L ANOS group was lower than other concentration groups(P<0.05).Compared with other time groups, the expression of MRP mRNA and MDR1mRNA on 24 hours after being transfected is the lowest(P<0.05).⑶Comparedwith 0.5μmol/L concentration group and control group ,the growth of cell wasrestrained when used ANOS at the concentration of 1.0μmol/L (P<0.05), onthe other hand, the growth of cell was not restrained when the concentration ofLipofectamine is 25μl/L.Conclusion: ⑴The highest dosage of radiation to Tca8113 cell line is 20Gy,cellwill die when the dosage is higher than 20Gy. ⑵ANOS can reduce theexpression of MRP mRNA and MDR1 mRNA,and partly reverse theradiation-resistance of human tongue carcinoma cell line(Tca8113).There iscorrelation between the recovery of radiation-resistant and ANOS transfection. ⑶The effect of ANOS depend on the time and concentration in cell. ⑷ANOS andLipofectamine are safe to human tongue carcinoma cell line(Tca8113)if theconcentration is lower than concentration of 1.0 μmol/L and 25 μL/mL(lipofectamine), respectively.