Proteomics Research The Influence Of SiRNA-ZNF139in Gastric Cancer Orthotopic Transplantation Nude Mouse Model

Objective: Gastric cancer is one of the most common malignant tumor inthe world, with a high fatality rate, is the most common digestive systemmalignant tumor. According to our research the abnormal expression ofZNF139is closely related to the occurrence of gastric cancer and the invasionand metastasis and the proliferation and apoptosis. Our experiment is conductto explore the influence of the ZNF139in the gastric cancer nude mousemodel which induced by orthotopic transplantation SGC7901gastric cancercell line. Using the two-dimensional fluorescence difference gelelectrophoresis (2D-DIGE) and LC/liquid (LC-MS) methods to study theeffects of inhibiting ZNF139on the primary and metastases of gastric cancer,the study will further provide the regulatory mechanism of ZNF139involvedin gastric cancer metastasis.Method：1Synthesizing of ZNF139small interfering RNA (siRNA-ZNF139) andtransfer the siRNA-ZNF139to gastric cancer cell line SGC7901(moderatelydifferentiated adenocarcinoma). The screening was conducted with G418aftertransfection.In vitro cultivation of SGC7901cells in logarithmic growth phase wasinterference with siRNA-ZNF139. qRT-PCR was used to detect the inhibitionrate of the siRNA-plasmid ZNF139at different concentrations. The optimalinterference cell line was obtained with the selection of the G418.2The establish of gastric cancer orthotopic transplantation of nude micemodel.The siRNA-ZNF139positive plasmid was select by G418and was usedto transfect the gastric cancer cells, the siRNA-ZNF139negative plasmidtransfect the gastric cancer was used as negative group, and the SGC-7901 without treatment as blank group.The stable transplanted tumor was obtained after six generationsubcutaneous culture. The stable tumor was attached to the stomach subserouswith biological glue in nude mice. In situ measuring of the transplant tumordiameter was conducted every4days. The mice was sacrifice with neckdislocation and the orthotopic transplantation tumor, abdominal swellinglymph nodes were collected at the diameter of the tumor is about1.0cm.Triplicate each sample, two samples stored in liquid nitrogen and the other onewas fixed with formalin.3Using two-dimensional fluorescent difference gel electrophoresis(2D-DIGE) technique to determine the protein expression before and after theinterference of siRNA-ZNF139in the stomach orthotopic transplantationtumor and abdominal cavity metastasis lymph nodes. Ettan spot picker,parallel gel enzyme solution and liquid application of LC/mass spectrometry(LC-MS) technology was used to extract protein for protein identification.4Western blot was used to check the protein expression validation ofnude mouse orthotopic transplantation tumor tissue and peritoneal metastasislymph node tissue.Result：1ZNF139expression after SiRNA-ZNF139transfect the gastric cancercell SGC7901line.qRT-PCR assay shown in the0μg/μl、0.4μg/μl、0.6μg/μl、0.8μg/μl、1.0μg/μl recombinant plasmid of SGC7901cells after transfection ZNF139relative expression of mRNA were1.0020±0.0765，0.6788±0.1032，0.3691±0.0242，0.1711±0.0133，0.2880±0.0506. The statistical analysisshowed that each group has significant difference (P<0.01), of which0.8μg/μlgroup inhibition efficiency is the highest with a inhibition rate of82.92%.2Morphological characters of the tumor after the siRNA inhibit ZNF139expression.After six generation of subcutaneous transplantation, tumor with thediameter of about1.0cm was harvested and planted in situ transplantation with OB biological adhesive method. About a week later all nude mice in theblank group and negative group abdominal tumors can be reached. At abouttwo weeks tumor can be reached in siRNA-ZNF139group and tumorcontinues to growth. Measurement of the diameter and the radius was conductevery4days and volume was calculated according to V=ab~2/2(a is the lengthto diameter, b for short radius). The blank tumor volume is slightly larger thanthat of the negative group, however the difference has no statistical difference(P>0.05). Compared with the negative group and blank group, theexperimental group tumor volume is significantly small (P <0.05).3The effects of siRNA inhibit ZNF139expression on the growth of nudemice celiac metastasis lymph nodes.The blank group, negative group and experiment groups were foundabdominal swollen lymph nodes. In the blank group the abdominal cavitylymph node metastasis rate was36.67%, and in the negative group the lymphnode metastasis rate was63.33%. The lymph node metastasis rate inexperiment group was66.67%.Compare the blank group and negative group the cavity lymph nodemetastasis rates have no significant difference (P>0.05), whereas theexperiment group has a significant low cavity lymph node metastasis rate andthe difference has statics difference (P<0.05).4Protein integrity analysis results.Protein extracted from the orthotopic transplantation tumor andabdominal cavity metastasis lymph nodes were separated by SDS-PAGEgel. The protein integrity was analysis by coomassie brilliant blue staining, theresults show that in situ transplantation tumor protein and metastasis lymphnodes have no obvious degradation.5The results of2D-DIGE and LC-MS.The protein isolated point of orthotopic transplantation tumor on the2D-DIGE map has an average of7658±47, having a matching rate of91.6%;the protein from the metastasis lymph node has an average of3117±82, andthe matching rate is92.5%. The results show that two-dimensional electrophoresis technology has a high repeatability. Use DeCyder DifferentialAnalysis Software to select9different proteins points on the2D-DIGE map ofthe orthotopic transplantation tumor. The analysis of protein was conductedvia LC-MS and five proteins were recognized and three was select afterfunctional analysis which may be related with gastric cancer protein. The threeproteins are Fascin, ANXA1and hnRNPA2/B1, the expression of Fascin andhnRNPA2/B1was down-regulated and siRNA-ZNF139was up-regulated ingastric cancer tissue. Five proteins were select by protein analysis inmetastasis lymph nodes, three kinds of protein were recognized via LC-MSand one may related to gastric cancer after function analysis. The protein isANXA5, and it has a low expression in siRNA-ZNF139gastric cancer group.6Western blot was used to confirm the Fascin, ANXA1and hnRNPexpression in situ transplantation tumor group and ANXA5, ZNF139expression in metastasis lymph nodes. The statistically analysis shows thatFascin, hnRNPA2/B1and ZNF139in sit transplantation tumor group has asignificantly down expression compare to the control group (P<0.01), whileANXA1expression increased significantly (P<0.01). Western plot result fromthe abdominal cavity lymph node shows ANXA5has a significantly lowexpression (P<0.01) in siRNA-ZNF139group compared with the controlgroup. These results are same as proteomics results.Conclusions:1Gastric cancer biological behavior, such as proliferation, invasion andmetastasis in human body can be simulated in nude mice. The establishmentof tumor model is simple and in high efficiency, which is a stable and reliableexperiment tool of gastric cancer research.22D-DIGE can perform statistical analysis for each protein points, whichcan ensure accurate, reliable and good repeatability. Combine with LC-MStechnology can accurately identify differences between proteins, analysis andidentification of protein structure.3ZNF139as a transcription regulatory factor may up-regulate theexpression of Fascin, hnRNPA2/B1and down-regulate the expression of ANXA1to promote tumor invasion and metastasis. In addition, the ZNF139may related to the proliferation, apoptosis, invasion and metastasis of gastriccancer.4The low expression of ANXA5in abdominal lymph nodes insiRNA-ZNF139group shows that in the process of metastasis, ZNF139mayinteract with ANXA5directly or indirectly promote the invasion andmetastasis of gastric cancer in the lymph nodes.