| Background Gastric cancer is one of the most common malignant tumor of digestive tract, chemotherapy in the treatment of gastric cancer has an important role. However, due to multi-drug resistance (multidrug resistance, MDR) generation, MDR is one of the reasons to treatment failure by chemotherapy. Therefore, To study gastric cancer MDR mechanism in order to enhance tumor chemotherapy and to develope a wider range of prospect for treating cancer. The protein is the function main performer of the vital activity and the function in the cell, therefore to study the MDR mechanism and to search the protein overall level to bear the medicine related protein member, It is very important for reversing tumor cell to reveal the MDR mechanism. Post-genomic era of proteomic as an important part of the study, providing a new platform for the life science research, while tumor MRD studies have brought new ideas and research in the field, it will possible bring a breakthrough for tumor's MRD. Application of proteomics technology researches group differences in human gastric cancer cell line SGC7901, and doxorubicin resistant cell SGC7901/ADR, and some of the differentially expressed proteins were identified mass spectrometry, high performance liquid chromatography electrospray ionization tandem mass spectrometry method for MS/MS analysis. The result showed that S100A11 and Pinl was highly expressed in SGC7901 cells, but expression was significantly reduced in SGC7901/ADR. To further confirm the expression differences, the study used the immunocytochemistry,Western-blot techniques and RT-PCR to detect S100A11,Pinl in SGC7901 and SGC7901/ADR. In order to further study the relationship between S100A11,Pinl and gastric cancer MDR,and to lay the foundation for gene,fection and RNA interference.Objective S100A11,Pinl in human gastric cancer cell line SGC7901 and SGC7901/ADR express differently, to analysis and explore its relationship with gastric cancer MDR relationship, and basis for gene transfection and RNA interference.Methods Human gastric cancer SGC7901 cells and SGC7901/ADR cells were inoculated on cover slips placed in 6-well plates in normal culture overnight, Immunohistochemistry Max Visin TM method staining S100A11 and Pinl in the expression of two difference cell lines, S100A11 polyclonal rabbit anti-human antibody, Pinl rabbit polyclonal antibody against human (U.S. ProteinTech Group, Inc, Max VisinTM purchase of Fuzhou New Biotechnology Development Co Ltd). rabbit polyclonal anti-human S100A11 antibody 1:100 dilution; rabbit anti-human Pinl polyclonal antibody 1:50 dilution. The total cellular protein extracted with RIPA lysis buffer and measured by Coomassie brilliant blue method of protein concentration, The total RNA were extracted by RNAiso Reagent lysates from two kinds of cells, using Western-blot and RT-PCR, respectively, from the protein level and RNA level of S100A11 and Pinl, detecting and analysising the expression level in SGC7901and SGC7901/ADR, To verify the mass spectrometry results and to further explore S100A11 and Pinl in the multidrug resistance of gastric cancer and tumor-related mechanisms of MDR may occur.Results 1. Immunocytochemistry detected S100A11,Pinl in SGC7901 cells over-expressed. S100A11 cytoplasm dark brown stained yellow-brown color, stain strongly positive (+++), in SGC7901/ADR cells significantly down-regulated expression of cytoplasmic yellow, stain weakly positive (+); Pinl cytoplasmic and Nucleus coloring, increase in the SGC7901 cell expression, dark brown stained yellow-brown color, strong positive staining for the expression (+++), down in SGC7901/ADR cells, staining was faint yellow, weakly positive (+).2. Western-blot Extraction of total cellular protein in SGC7901 and SGC7901/ADR, SGC7901 cell total protein concentration 3.5ug/ul, SGC7901/ADR total cellular protein concentration 3.2ug/ul. In SGC7901 cells the mean protein of S100A11 was 0.69±0.13, In SGC7901/ADR was 0.22±0.08; In SGC7901 cells the mean protein of Pinl was 0.81±0.15, in SGC7901/ADR cells the mean protein was 0.52±0.09, P<0.05, difference was significant.3. RT-PCR results:in SGC7901 and in SGC7901/ADR cells band signal strength of S100A11 was similar and no significant difference; In SGC7901 and SGC7901/ADR cells Pinl was no significant differences, the mean mRNA of S100All in SGC7901 cells was 0.60±0.87, in SGC7901/ADR cells was 0.59±0.78; In SGC7901 the mean mRNA of Pinl was 1.49±0.13, in SGC7901/ADR was 1.41±0.16, P>0.05, the difference was no significant.Conclusions 1. At the protein level, the expression of S100A11and Pinl in human gastric cancer cell line SGC7901 and SGC7901/ADR was resistantly different, both over-expressed in SGC7901, and the expression in SGC7901/ADR cells was reduced.2. At the mRNA level, S100A11,Pinl in SGC7901 and SGC7901/ADR cells had no significant difference. The different expression of S100Alland Pinl was not increased synthesis of SGC7901 or decreased synthesis of SGC7901/ADR, but the synthesized events may be related to S100A11 and Pin1 protein degradation to reduce in SGC7901 cells or the degradation to increase in SGC7901/ADR cells.3.The different expression of S100All and Pinl in SGC7901 and SGC7901/ADR may be related with gastric cancer MDR. |
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