| Objective:1.To study the relationship between lipid rafts and drug resistance.Flotillins,a functional protein on the lipid raft,was selected,to study the reversal of resistance.2.In order to investigate the mechanism of drug resistance in tumor drug-resistant cells after Flotillins knockdown,the expressions of ERK1/2,p-ERK1/2 in MAPK pathway and IRS-1 and PI3K in PI3K/Akt pathway were detected.Methods:1.Two human tumor cell lines SMMC-7721 and HCT-15 were pulsed with high-concentration doxorubicin(ADM)for a long period of time and the half-inhibitory concentration(IC50)was calculated by assessing the sensitivity of ADM at different concentrations by the Cell Titer 96R Aqueous One Solution(MTS).The cell proliferation inhibition rate was calculated as follows:cell proliferation inhibition rate=1-(OD in the experimental group-OD in the blank control group)/(OD in the negative control group-OD in the blank control group);then,the half--maximal inhibitory concentration of the drug(IC50)was estimated by the SPSS software.2.Three shRNA sequences(sh1,sh2,sh3)and one negative control(Con)were designed and synthesized based on the human Flotillin-1 and Flotillin-2 genes.After annealing,the lentivirus was digested by T4 ligase The vector was transformed into E.coli DH5a and sequenced to obtain the recombinant plasmids.The recombinant plasmids were co-transfected into 293ft cells with helper plasmids(PSPA,PMD).The supernatant was used to infect SMMC-7721/ADM and HCT-15/ADM cells and the transfection efficiency was observed.RT-PCR and Western blot were used to detected of Flotillin-1 and Flot-2 mRNA transcription and protein expression levels,screening out the best target.3.The expression of flotillin-1 and flotillin-2 in parental cells and resistant cells was detected by Western blot.4.The IC50 and RI of each group of cells were calculated by SPSS(the calculation method is as follows 1),to observe the rule of drug resistance change of tumor drug-resistant cells after flotillin-1 or flotillin-2 knockdown.Flow cytometry was used to detect the cell apoptosis and cell cycle.5.The expressions of ERK1/2,p-ERK1/2 in MAPK pathway and IRS-1 and PI3K in PI3K/Akt pathway were detected by Western blot,and the mechanism of drug resistance reversal in tumor drug-resistant cells after flotillin-1 or flotillin-2 knockdown was understood.Results:1.Two tumor drug-resistant cell lines were obtained after induction by ADM:SMMC-7721/ADM and HCT-15/ADM.The ADM IC50 values in the SMMC7721 and SMMC7721/ADM cells were 0.219±0.035 and 3.995±0.144 ?g/mL,respectively,and the resistance index of the SMMC7721/ADM cells was 18.24.The ADM IC50 values of the HCT-15 and HCT-15/ADM cells were 0.2±0.013 ?g/mL and 3.516±0.26 ?g/ml,respectively,and the resistance index of the HCT-15/ADM cells was 17.58.(High resistance is resistance index greater than 15)2.After Flotillin-1 and Flotillin-2 knockdown,the mRNA and protein expression levels were dramatically reduced respectively.All sequences at the mRNA levels were effective compared with the control.Then,screening out the best target for Flotillin-1 and Flotillin-2,respectively,and protein levels were detected.3.The expression levels of Flotillin-1 and Flotillin-2 are significantly higher than those in the parental cell lines.4.After Flotillin-1 knockdown,the sensitivity to ADM was significantly rasied.In the SMMC7721/ADM and HCT-15/ADM cells,the IC50 values to ADM were decreased to 2.385±0.162 and 2.507±10.026?g/ml,meanwhile,the reversal levels of resistance to ADM were 1.65-fold and 1.38-fold respectively;After Flotillin-2 knockdown,the sensitivity to ADM was significantly rasied.In the SMMC7721/ADM and HCT-15/ADM cells,the IC50 values to ADM were decreased to 1.50810.109 and 1.57±0.07?g/ml,meanwhile,the reversal levels of resistance to ADM were 2.61-fold and 2.2-fold respectively.Knockdown of flotillin-2 promoted apoptosis,and knockdown of flotillin-1 had no significant influence.Downregulation of flotillins expression can induce significant cell cycle arrest.5.Knockdown of flotillin-1 reduced the expression levels of phosphorylated ERK1/2 in resistant cells,compared with that in the control group;however,knockdown of flotillin-2 induced up-regulation.The IRS-1 and PI3K levels in the Flotl-RNAi and Flot2-RNAi groups were dramatically down-regulated compared with those in the control group.Conclusions:1.After the knockdown of flotillin-1 and flotillin-2 in resistant cells,cell sensitivity to ADM increased,IC50 decreased,cell resistance decreased,and drug resistance was reversed.The drug resistance reversal index of drug-resistant cells was higher after Flotillin-2 knockdown.2.Knockdown of flotillin-1 reduced the expression levels of phosphorylated ERK1/2 in resistant cells,compared with that in the control group(Con group);however,knockdown of flotillin-2 induced up-regulation,the results assessed ERK1/2 pathway produce an not effect on the decrease of resistance after lipid rafts destruction.3.The levels of IRS-1 and PI3K proteins in the Flotl-RNAi and Flot2-RNAi groups were significantly down-regulated,the results assessed that the change of drug resistance after lipid raft destruction may be related to the PI3K/AKT signaling pathway,but the influence of permeability and efflux on drug resistance cannot be excluded. |
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