Established Cell Lines That Down-regulation Of S100A6Expression And Correlation Of S100A6to Multidrug Resistance Of Human Gastric Cancer Cell Line SGC7901

Background： Gastric cancer is one of the most common malignant tumors, whichmainly treated by surgery and systemic therapy regiments in clinical. Systemic therapyregiments especially chemotherapy, has an important role during the treantment. It caneffectively inhibit the resurgence of primary lesions and can also eliminate the distancemetastasis, has important significant for patients’ prognosis and long-term survival.However, the effect of clinical gastric cancer chemotherapy is far from ideal, the keyfactor is the generation of multidrug resistance. Therefore further study the mechanismof gastric cancer multidrug resistance is the current focus in the study of gastric cancercurrently.In order to find out new MDR related proteins in gastric cancer, Proteomicstechnology was used to compare the differences between the human gastric cancer cellline SGC7901, its Adriamycin-resistant cell line SGC7901/ADR and the reversed cellline by Salvia miltiochiga. Then the differential proteins among the three cell lines wereindentified by HPLC-ESIMS/MS. The results showed that S100A6is highly expressedin SGC7901, as well as the revearsed cell by Salvia miltiochiga, and down-regulated inSGC7901/ADR. In order to verify the experimental results, we usedimmunocytochemmistry, Western-blot analysis to detect S100A6expression levelbetween SGC7901and SGC7901/ADR cells, the results were verified this differencein expression. However, by using RT-PCR, we found that, there was no significantdifference between SGC7901and SGC7901/ADR cells in S100A6mRNA expression. In order to engage further study on the relationship between S100A6and MDR ofgastric cancer, we constructed the human S100A6gene shRNA eukaryotic expressionplasmid and found out the most effective RNA interference fragment.Objective: Established cell lines that down-regulated expression of S100A6by RNAinterference technology on purpose of further research about the relationship betweenS100A6and MDR of gastric cancer.Methods: Enlarged shRNA-1, shRNA-3, NC plasmid by using competence DH5α E.coli as a carrier. Cultured the SGC7901cell line, selected the best concentration forG418screening test. SGC7901cells were transfected with shRNA-1, shRNA-3or NCplasmid by liposome transfection, and then cultured these transfected cells with G418for40days in order to get the stable transfection cells. SGC7901cells, SGC7901/ADRcells and these three stable transfection cells were inoculated on cover slips placed in6-well plates in normal culture. Using Immunocytochemical staining method detectedthe S100A6protein expression in these five cell lines, then compared the differencesbetween the protein expressions of them. Cultured these five kinds cell lines, extractedthe total cellular protein from the five kinds of cells and measured their concentration;cultured these five kinds cell lines as well, extracted the total cellular mRNA from thefive kinds of cells and measured their concentration. Then using western-blot andRT-PCR detected the S100A6expression in protein level and mRNA level. CCK8experiment, the five kinds of cells were inoculated in96-well plates with the density of1×104/ml and every well100ul, and then divided each cell into7groups, every grouphad5repeated wells, cultured those cells for24h. Perpared cell-culture mediums of sixgradients (contained ADR), using these six kinds of cell-culture mediums and normalcell-culture to culture the cells for48h. Toke out the plates and mixed with cck810ulper well, waited for4h. Every well was detected by spectrophotometry at450nm and wecan get the IC50and RI of each kind of these five cell lines.Results:1. Established three kinds of cell lines: shRNA-1stable transfection cell line,shRNA-3stable transfection cell line, NC stable transfection cell line.2.Immunocytochemistry results: immunocytochemistry detected S100A6in SGC7901 cells and NC stable transfection cells over-expressed. S100A6cytoplasm dark brownstained yellow-brown color, strain strongly positive (+++), in shRNA-1stabletransfection cells, shRNA-3stable transfection cells and SGC7901/ADR cellssignificantly down-regulated expression with pale yellow cytoplasm, stain weaklypositive (+).3.Western-blot result: In SGC7901cells the mean expression of S100A6protein was0.97±0.08, in NC stable transfection cells was0.93±0.07, inSGC7901/ADRcells was0.78±0.03, in shRNA-1stable transfection cells was0.68±0.03, in shRNA-3stable transfection cells was0.74±0.05. According to One-Way ANOVA analysis,SGC7901cells compared with SGC7901/ADR cells, shRNA-1stable transfection cellsor shRNA-3stable transfection cells, P<0.05, the difference was significant. SGC7901cells compared with NC stable transfection cells, P>0.05, the difference was notsignificant.4. RT-PCR result: In SGC7901cells the mean expression of S100A6mRNAwas0.975±0.006, in NC stable transfection cells was0.961±0.034, in SGC7901/ADRcells was0.935±0.027, in shRNA-1stable transfection cells was0.768±0.088, inshRNA-3stable transfection cells was0.806±0.055. According to One-Way ANOVAanalysis, SGC7901cells compared with SGC7901/ADR cells or NC stable transfectioncells, P>0.05, the difference was not significant. SGC7901cells compared withshRNA-1stable transfection cells or shRNA-3stable transfection cells, P<0.05, thedifference was significant.5.CCK8test result：Under the influence of ADR the IC50ofSGC7901cells was0.336±0.045ug/ml, the IC50of NC stable transfection cells was0.342±0.041ug/ml, the IC50of SGC7901/ADR cells was1.288±0.168ug/ml, the IC50ofshRNA-1stable transfection cells was0.564±0.084ug/ml, the IC50of shRNA-3stabletransfection cells was0.402±0.001ug/ml. According to One-Way ANOVA analysis,SGC7901/ADR cells compared with the other four kinds of cells, P<0.05, the differencewas significant. SGC7901cells compared with shRNA-1stable transfection cells,P<0.05, the difference was significant. SGC7901cells compared with shRNA-3stabletransfection cells or NC stable transfection cells, P>0.05, the difference was notsignificant.Conclusions:1. shRNA-1eukaryotic expression plasmid is the best interference target plasmid in our experiment, it could interfere the expression of S100A6statistically inSGC7901cell line.2. Eukaryotic expression plasmids expressing shRNA sectionstargeting human S100A6gene could stable transfection SGC7901cells, and inhibit theexpression of S100A6gene. Established cell lines that own-regulated expression ofS100A6.3. S100A6protein could contribute to gastric cancer MDR.