| Backgroud:Gastric cancer is one of the most common malignant tumors.Early gastric cancer generally has no obvious clinical symptoms,once diagnosed,usually belong to II?III period,chemotherapy plays an important role in the comprehensive treatment of advanced gastric cancer.Although great progress has been made in the treatment of chemotherapy regimens and chemotherapeutics,the overall prognosis is poor,the main reason is multidrug resistance of tumor cells,so that the chemotherapy of majority of patients with gastric cancer failed.Therefore,it is one of the hot spots in the research of gastric cancer to study the mechanism of multidrug resistance of gastric cancer in order to search for reversal agents with the high efficiency and low toxicity.Sh RNA-1 stable transfection cell line,sh RNA-3 stable transfection cell line and negative control NC stable transfection cell line are our subject group by taking the human gastric cancer cell line SGC7901 to established.We use these cell lines,SGC7901 cells and Adriamycin-resistant cell line SGC7901/ADR as the research objects.We compared them by Immuncytochemistry,Western blotting,RT-PCR and MTT to find that S100A6 low expression can reduce the multidrug resistance of gastric cancer cells to a certain extent and S100A6 may have a positive correlation with ?-catenin.In order to further study the relationship between?-catenin and the mechanism of MDR in gastric cancer,we will establish low?-catenin expressed sh RNA transfection cell lines to study whether the down-regulation of ?-catenin can reverse the multidrug resistance of gastric cancer toa certain extent.Objective: In this study,human gastric cancer cell line SGC7901/ADR as the research object,we use RNA interference technology to transfected SGC7901/ADR cells to establish low ?-catenin expressed cell lines in order to further study whether the down –regulation of ?-catenin could reverse the multidrug resistance of gastric cancer to a certain extent.Methods:Using liposome transfection transfect SGC7901/ADR cells to establish sh RNA-CTNNB1 and sh RNA-NC cells,culturing SGC7901 cells ? SGC7901/ADR cells and two transfection cells,and extracted the total cellular protein and the total cellular m RNA from the four kinds of cells.Then we detect the ?-catenin expression in protein level and m RNA level by Western blotting and RT-PCR.We inoculate the four kinds of cells in 96-well plates to do CCK8 experiment for determination of OD per hole at 450 nm,and then calculate their IC50 and RI.Results: 1.Established two kinds of transfection cell lines:one is sh RNA-CTNNB1 transfection cell line which down-regulation ?-catenin and the other one is negative control sh RNA-NC transfection cell line.2.Western blotting result:The average expression of ?-catenin protein in SGC7901 cells ? SGC7901/ADR cells ?sh RNA-CTNNB1 transfection cells and sh RNA-NC transfection cells was0.434±0.244 ? 1.372±0.264 ? 0.661±0.083 ? 1.436±0.178.According to independent-samples T test,SGC7901/ADR cells compared with SGC7901 cells?sh RNA-CTNNB1 transfection cells,P<0.05,the difference was significant.SGC7901/ADR cells compared with sh RNA-NC cells,P>0.05,the difference between two kinds of cells was not significant.SGC7901 cells compared with sh RNA-CTNNB1 transfection cells,P>0.05,the difference was not significant between them.3.RT-PCR result: The average expression of ?-catenin m RNA in SGC7901 cells ? SGC7901/ADR cells ? sh RNA-CTNNB1 transfection cells and sh RNA-NC transfection cells was 0.659±0.05 ? 1.106±0.043 ? 0.747±0.027 ?1.111±0.043.According to independent-samples T test,SGC7901/ADR cells compared with SGC7901 cells?sh RNA-CTNNB1 transfection cells,P<0.05,the difference was significant.SGC7901/ADR cells compared with sh RNA-NC transfection cells,thedifference between two kinds of cells was not significant.SGC7901 cells compared with sh RNA-CTNNB1 transfection cells,P>0.05,the difference was not significant.4.CCK8 test result:Under the action of Adriamycin,the IC50 of SGC7901 cells was 0.378±0.026ug/ml,the IC50 of SGC7901/ADR cells was1.371±0.044ug/ml,the IC50 of sh RNA-CTNNB1 transfection cells was0.497±0.017ug/ml,the IC50 of sh RNA-NC transfection cells was1.414±0.079ug/ml.According to independent-samples T test,SGC7901/ADR cells compared with SGC7901 cells ? sh RNA-CTNNB1 transfection cells,P<0.05,the difference was significant.SGC7901/ADR cells compared with sh RNA-NC transfection cells,P>0.05,the difference between two kinds of cells was not significant.We compare SGC7901 cells with sh RNA-CTNNB1 cells to find P<0.05,the difference was significant.Conclusions:.1.Recombinant plasmid expression vector p GPU6/GFP/Neo-sh RNA-CTNNB1 could transfect SGC7901/ADR cells by targeting human ?-catenin gene,and specially inhibit the expression of ?-catenin gene.2.?-catenin protein may contribute to gastric cancer MDR. |
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