Reversal Of Multidrug Resistance On Stomach Cancer Cell SGC7901/Mdr1by ShRNA Targeting Mdr1

Backgroundmdrl expression and the effect of chemotherapy of gastric cancer is closely related, multidrug resistance of gastric cancer (MDR) also need to continue to explore and need more relatively homogeneous biological characteristics, resistance stability in gastric cancer MDR cell research. At the respect of a single biological properties and resistance stability, establish MDR cells using genetic methods of biological transfer is more advantage than that using drug-induced formation. Many gastric cancer MDR cells used for the study are drug-induced formation, and we have established SGC7901/mdrl cell line through transfecting the the mdrl cDNA into SGC7901cells. However, the causal relationship between expression of mdrl and the MDR in mdrl SGC7901/mdrl cells need to be confirmed.ObjectiveShRNA targeting mdrl expression plasmids were stably transfected into the gastric cancer SGC7901/mdrl cells, observe changes of mdrl expression and resistance, determine whether the drug resistance of gastric cancer cells SGC7901/mdrl was caused by the overexpression of mdrl and assistant the mdrl-induced MDR research platform that use the SGC7901/mdrl cells as the research subject.Methods1. Cell culture:The SGC7901and SGC7901/mdrl were cultured conventionally. Those cells transfected with empty vectors, mdrlshRNA-3266, and mdrl shRNA-2631cells were cultured in RPMI1640medium supplemented with300μg/mlG418to maintain the screening.2. Amplification and extraction of the plasmid:The the competent DH5a cells containing pSUPER-EGFP, or pSUPER-EGFP-mdr1shRNA-326or pSUPER-EGFP-mdr1shRNA-2631were cultured and the plasmids were extracted using plasmid extraction kit.3. Transfection of plasmid and selection:The empty vector, mdr1shRNA-326, and mdr shRNA-263were transfected into SGC7901/mdr1cells using lipofectamineTM2000. The blank group was added PBS. After48h of transfection, G418was added into the medium in600μg/ml of the final concentration and the expression of green fluorescent protein was determined using confocal microscopy to jurdge the screening effect.4. Selection of cells:The cells of each group were collected after the drug selection and the cells expressing green fluorescent protein were selected using fluorescence-activated cell sorting (FACS). The cells of each group were named mdrl shRNA-326stability group, mdr1shRNA-of2631stability group and empty plasmid stability group. The SGC7901and SGC7901/mdr1cells were used as the parental control and blank control, respectively.5. Detection of mdr1:The mdr1mRNA and protein expression of P-gp were detected by RT-PCR and Western blot analysis respectively, β-actin was included as the control.6. Intracellular ADR accumulation:Flow cytometry was used to detect specific fluorescence intensity of ADR within the cells in each group after treatment of10μg/ml of ADR for90min. PBS was instead of the ADR in the blank control.7. Detection of sensitivity to ADR:The cells of each group were treated by ADR in the final concentration of1.65μg/ml,3.3μg/ml,33μg/ml,330μg/ml,660μg/ml for48h, suppression rate and IC50were determined using the MTT assay in each group, the PBS instead.of the ADR and cell-free were used as a blank control and reagent control.8. Statistical analysis:The SPSS17.0statistical software was used to process the data. The numerical variable data were expressed with (x±s), ANOVA were used to compare these data. The positive rate was calculated with count information.Results1. Extraction of the plasmid:The plasmid of pSUPER-EGFP-mdr1sh326, pSUPER-EGFP-mdrlsh2631and pSUPER-EGFP were extracted. The concentration of all the plasmids is between0.2～0.4μg/ul, the range of A260/280is1.7～1.9. From the agarose electrophoresis map, we can see the supercoiled bands are clear and brightness.2. Selection of cells:Each group of transfected cells were treated by G418for7days and the cells of blank control were fully killed. The green fluorescent protein-positive rates in the other groups are not high. After four weeks of G418treatment, the green fluorescent positive cells of each group were selected by flow cytometry and the rates of each groups are98.7%,96%, and95.0%, respectively.3. shRNA interferes mdrl mRNA expression in SGC7901/mdrl cells:RT-PCR results show that two kinds of shRNA can effectively inhibit mdrl mRNA expression in SGC7901/mdrl cells, the mdrl mRNA level in mdrlshRNA-2631cells is lower than that in mdrlshRNA-326cells.4. shRNA inhibits P-gp expression in SGC7901/mdrl cells:Western Blot results shows that the P-gp expression level in mdrlshRNA-326stability group and mdrlshRNA-2631stability group are lower than that in empty plasmid group and SGC7901/mdrl group, and close to the the SGC7901group.5. The effect of mdrl shRNA on the ADR accumulation:The ADR accumulation in mdrl shRNA-326group and mdrl shRNA-2631group were higher than that in SGC7901/mdrl group and the empty plasmid group (P<0.05), but lower than that in the SGC7901group (P<0.05).6. The effect of mdrl shRNA on the sensitivity to ADR:The sensitivity to ADR in mdrl shRNA-326group and mdrl shRNA-2631group were higher than that in SGC7901/mdrl group and the empty plasmid group (P<0.05), but no significance compare with that in the SGC7901group (P<0.05).ConclusionshRNA targeting mdrl can effectively reverse the resistance in SGC7901/mdrl cells after inhibiting the expression of mdrl and high expression of P-gp in SGC7901/mdrl cells leads to the multidrug resistance.