Screening And Identification Serum Differential Protein Of Human Gastric Cancer Orthotopic Transplantation Nude Mouse Model Before And After Sirna Inhibit ZNF139Expression

Objective: Orthotopic transplantation nude mouse model was establishedwith gastric cancer SGC7901cell line. Two-dimensional difference gelelectrophoresis（2D-DIGE） and liquid chromatography-mass spectrometry(LC-MS) were used to study the inhibition effect of ZNF139on serum ofgastric cancer orthotopic transplantation in nude mice.This study willprovide the information about ZNF139involved in gastric cancer and furtherclues for discovering new tumor markers.Method：1Gastric cancer cell line SGC7901was cultivated, siRNA was designedaccording to the ZNF139gene sequence，siRNA-ZNF139expression plasmidswere constructed. The recombinant plasmid was transfected into SGC7901cells by X-tremeGENE HP transfection reagent. The expression of theZNF139mRNA in the transfected cells was evaluated by qRT-PCR and theinhibition efficiency was calculated according to the results.2The recombinant plasmid of siRNA-ZNF139and negative plasmidwere transfected into gastric cancer SGC7901cells, stable transfection cellswas screened by G418.The stable transfected cells and ordinary SGC7901cells were injected into nude mouse subcutaneous. The subcutaneous tumorsize were measured every4days, the subcutaneous tumor was cut into1cubicmillimeter tumor block when it reached about1.0centimeter. The tumor blockwas repeatedly subcutaneously inoculated between nude mice, and the sixthgeneration tumor block was transplanted on the stomach wall of nude mice byimproved OB biological glue method.3High abundance proteins in the serum of orthotopic transplantationnude mouse of human gastric cancer were removed by albumin /immunoglobulin clear kit.The change of serum protein and its integrity wasdetected by SDS-PAGE.4The proteins in nude mouse serum samples were separated byfluorescence two-dimensional difference gel electrophoresis. Interestingproteins were selected by DeCyder Differential Analysis Software and werepicked out use ettan spot picker.The difference proteins were identified byliquid chromatography-mass spectrometry (LC-MS) after in-gel digest. TheSEQUEST computing method in BioWorks software was applied for databaseretrieval.5Parts of proteins which were identified were evaluated by Western blotto measure the protein expression levels in order to test whether it maintainsconsistency with the proteomics test results.Result：1ZNF139mRNA expression in transfected SGC7901cells with differentconcentrations plasmid of siRNA-ZNF139The expression of the ZNF139mRNA is1.003±0.0970，0.7517±0.0436，0.3611±0.0445，0.1622±0.0181，0.5531±0.0377in transfected SGC7901cellsat the final concentration of0μg/ml,0.4ug/ml,0.6ug/ml,0.8ug/ml,1.0ug/ml detected by qRT-PCR. The difference has statistical significancecompared with0μg/ml (P<0.05), and the83.83%in0.8μg/ml group has thebest inhibition efficiency.2The effects of siRNA inhibit ZNF139on the growth of subcutaneoustumors in nude miceThe subcutaneous tumor can be observed in the blank group and negativegroup after6days inoculation and10days in the siRNA-ZNF139grouprespectively. The subcutaneous tumors become larger as time went on. Thesize of blank group subcutaneous tumors are slightly larger than negativegroup, however there are no statistical significance（P>0.05）. The volume ofsubcutaneous tumors in siRNA-ZNF139group are smaller than the negativecontrol group, there are statistical significance (P <0.05).3Morphological observation of orthotopic transplantation nude mouse gastric cancer modelOne nude mouse died of overdose of anesthesia in negative control group,and one mouse died in blank group and another in siRNA-ZNF139grouprespectively two days after operation.Two weeks later, cancer tissue could betouched epigastrium in the15nude mice and the cancer mass keeps increasing.The situ tumor volumes of siRNA-ZNF139group are smaller than that inblank control group and negative control group the difference has statisticsignificance (P<0.05). The orthotopic transplantation tumor was confirmedwith histopathological examination showstumor cells larger nucleu withhyperchromatism, the emerging rate of tumor is100%.4Purification of serum protein and integrity detectionThe serum protein which separate by SDS-PAGE have no obviouslydegradation by coomassie brilliant blue staining. The high abundance proteinsare obviously decreased and low abundance proteins are more apparent afterthe treatment of albumin/immunoglobulin clear kit.5Screening and identification of serum differential protein of humangastric cancer orthotopic transplantation nude mouse model before and aftersiRNA inhibit ZNF139expressionAbout4487±46proteins are separated by2D-DIGE and the matchingrate is83.1%. Seven differences proteins were selected with DeCyderDifferential Analysis Software selects. Five kinds of proteins are identified byLC–MS，which is α2-MG，KNG1，mTERF，DIXDC1and APOE. Amongthese proteins APOE and KNG1are down-regulate whileα2-MG, mTERFand DIXDC1up-regulating in siRNA-ZNF139group.6The effects of siRNA inhibit ZNF139on expression of APOE, KNG1,mTERF, ZNF139in serum of human gastric cancer orthotopic transplantationnude mouse modelThe result of western blot shows that APOE,KNG1and mTERF have nosignificant differences between blank group and negative group (P>0.05),APOE and KNG1are significantly down-regulated, while mTERF issignificantly up-regulated in siRNA-ZNF139group, the difference has statistical significance（P<0.05）. The result is also consistent with theproteomic result.Conclusions:1siRNA-ZNF139exprssion plasmid can be used to inhibit the expressionof ZNF139gene.2siRNA-ZNF139exprssion plasmid can inhibit the growth of subcutane-ous tumor and orthotopic tumor in nude mice model.3The orthotopic transplantation nude mouse model of human gastriccancer can be used to simulate the biological behavior of gastric cancer inhuman body, such as proliferation, invasion and metastasis. Further moresimple operation, high rate of tumor formation by improved OB biologicalglue method.It is easy for observation, measurement and interventions. So, atpresent, the model is considered to be a relatively ideal experimental model tostudy gastric cancer.4High abundance proteins, such as ibumin and immunoglobulin,removed from serumprovides the foundation for serum proteomics research.5DIGE with highly accuracy, repeatability and sensitivity can be used toaccurately identify proteins with the usage of LC-MS.6ZNF139as a regulation factor at the transcriptional level, may bepromote tumor development and progression through promoting APOE,KNG1and inhibiting mTERF expression.