| Part one The expression level and clinical significance of PIAS3 in gastric cancer and adjacent non-cancerous tissueObjective:To observe the expression level of PIAS3 in gastric carcinoma and adjacent normal tissues.To clarify the relationship between the expression of PIAS3 in tumor cells and the malignant behavior of gastric cancer.To investigate the significance of PIAS3 expression in the prognosis of gastric cancer patients.Methods:All patients were signed and agreed to participate in the study and approved by the local hospital ethics committee.Retrospective study of130 patients who have received radical gastrectomy surgery in our hospital from October 2005 to October 2010.All of these patients did not receive preoperative neoadjuvant chemotherapy,postoperative were treated with fluorouracil-based first-line chemotherapy for at least 6 cycles,the patients received the regular review every 3 months after operation and follow-up to died.Clinical examination of blood,urine,routine,liver and kidney function and tumor markers and other biochemical results were collected.In addition,preoperative ultrasound,abdominal CT,chest X-ray examination and endoscopy and other imaging findings should also be collected.The expression of PIAS3 and STAT3 protein in 130 cases of radical gastrectomy was detected by immunohistochemistry(IHC).Ten fresh gastric cancer samples and non-cancerous tissue samples were collected,The expression of PIAS3 and STAT3(P-STAT3)mRNA and protein were detected by real-time PCR and Western blot.Based on the expression of PIAS3 protein in immunohistochemistry,the clinical and pathological information were analyzed by SPSS18.0 software.Results:1 The general condition of patients with gastric cancer:the ratio of male to female patients is about 3.5:1,about 46.92%of patients under the age of 60,the pathological results showed that the serosal invasion of the tumor accounted for 84.62%,63.85%of patients with lymph node metastasis.2 Expression of PIAS3 and STAT3 proteins in tumor tissues of patients with gastric cancer:Immunohistochemical(IHC)results showed that the positive results of PIAS3 protein expression were mainly located in the cytoplasm,yellow or brown granules,95 cases of all gastric cancer tissues Low expression(73.08%),and 35%were high(26.92%).STAT3 protein expression was mainly located in the nucleus,yellow or brown granules,42cases of gastric cancer in 42 cases of low expression(the proportion of32.30%),88 cases of high expression(the proportion of 67.69%).There was a significant negative correlation between PIAS3 and STAT3 protein expression in tumor tissue(R=-0.656,P<0.001).3 Western blot analysis showed that the expression level of P-STAT3protein in tumor tissue was significantly higher than that in adjacent normal tissues(5.20±0.63 vs.1.00±0.36,P<0.05).The expression level of P-STAT3 protein in tumor tissue was about 5.2 times that of normal tissue.(P<0.05).The expression level of PIAS3 protein in tumor tissue was significantly lower than that in adjacent normal tissues(1.00±0.30 vs.3.15±0.43,P<0.05).The expression level of PIAS3 protein in tumor tissue was significantly lower than that in adjacent normal tissues.Real-time PCR results showed that the expression level of STAT3 mRNA in tumor tissues was 2.05±0.21(P<0.05),while PIAS3 mRNA was adjacent to the adjacent tissues The expression level in normal tissues was 2.48±0.43(P<0.05)in tumor tissue.4 The relationship between the prognosis of patients with gastric cancer and the level of PIAS3 expression:130 patients were followed up for more than 5 years(of which the shortest 12 months of survival,the longest 126months),the median survival time of patients was 37.5 months(95%confidence interval:18~79 months),in the last follow-up there is one patient survived.The relationship between the overall survival time,tumor location,size,differentiation,local infiltration,lymph node metastasis and PIAS3expression was analyzed.The decrease of PIAS3 expression was positively correlated with local infiltration and lymphatic metastasis of gastric cancer.PIAS3 protein expression in GC tissues(HR=4.801(2.892-7.970),P<0.001),status of lymph node metastasis(HR=1.725(1.152-2.584),P=0.008)and Lauren typing(HR=1.514(1.074-2.133),P=0.018)were identified as the independent factors of OS in all enrolled GC patients following the multivariate analysis(Cox regression with bootstrapping performance).Part two PIAS3 inhibits proliferation and metastasis of human gastric cancer cells by limiting STAT3 activationObjective:The expression of PIAS3 in gastric cancer cells was up-regulated or down-regulated by virus-infected gene modification technique.The proliferation and invasion of gastric cancer cells were observed and the intrinsic molecular mechanism of PIAS3 involved in the malignant biological behavior of gastric cancer cells was investigated.Methods:The human gastric cancer cell lines AGS,HGC-27,KATO-III,and human normal gastric mucosa cell line GES-1 were observed in vitro.Real-time PCR(Real-Time PCR)and western blot were used to detect the expression of STAT3 and PIAS3 mRNA and protein in each cell line.The virus-infected gene modification technique was used to construct the up-regulated or down-regulated PIAS3 gastric cancer cells The survival and proliferation ability of the cells were observed by MTT and EDU.The transwell was used to detect the migration ability of the cells.Results:1 Basal expression level of PIAS3/STAT3 in different Cell lines:The expression level of PIAS3 in normal gastric mucosa GES-1 cell was 2.75±0.13,meanwhile,it was 0.85±0.08 in AGS gastric cancer cell line and 0.98±0.11 in HGC-27 gastric cancer cell line,1.35±0.23 in KATO-III gastric cancer cell line,respectively.The expression level of P-STAT3 in GES-1 cells was 0.38±0.06,while it was 0.98±0.06 in AGS gastric cancer cell lines,0.67±0.02 in the HGC-27 gastric cancer cell line and 0.55±0.03 in the KATO-III gastric cancer cell line.Compared with normal gastric mucosal cell GES-1,PIAS3 expression decreased and STAT3 activation(P-STAT3)increased in gastric cancer cells.2 According to the basic expression level of PIAS3,a stable gastric cancer cell line was constructed.The results showed that the expression of STAT3 could significantly increase the activation level of STAT3 in gastric cancer cells.In vitro functional experiments showed that overexpression of PIAS3 significantly inhibited the proliferation of gastric cancer cells and the ability of invasion and migration,whereas the reverse expression of PIAS3significantly promoted the proliferation and invasion and migration of gastric cancer cells.Part three The antitumor effect of PIAS3 in vivo and its molecular mechanism in gastric cancer xenografts in nude miceObjective:BALB/c Nude nude mice subcutaneously transplanted tumor model was constructed by constructing gastric cancer cell line with stable overexpression of PIAS3.The effect of PIAS3 on tumor growth was observed by comparing the tumor growth rate.Methods:AGS-PCDH-PIAS3,a highly expressed gastric cancer cell line and AGS-PCDH-control were used to construct BALB/c Nude nude mice subcutaneous transplanted tumor modle.The expression of PIAS3,STAT3and Ki67 in tumor tissue was confirmed by immunohistochemistry.Furthermore,validating the role of PIAS3 in tumor growth.Results:1 The gastric cancer subcutaneous tumor-bearing mice general situation:The control group nude mice weight gradually decreased(35 days average body weight 14.3±5.2g),activity decreased,apathetic,the skin gradually grew tumor mass,however,AGS-PCDH-PIAS3 group Nude mice showed no progressive decrease in body weight(average body weight of 19.3±3.5g on the 35th day).The motor ability of the nude mice was acceptable.The surface of the skin grew gradually,and the growth of the tumor was slower than that of the control group.2 Overexpression of PIAS3 can inhibit the growth of tumor transplanted tumor:Compared with the transfected empty plasmid group,the tumor volume of AGS-PCDH-PIAS3 group(472.99±88.83mm~3)was significantly smaller than that of AGS-PCDH-control group(1748.39±204.39 mm~3),the difference was statistically significant.3 The expression of STAT3 in PIAS3 group was significantly decreased and the proliferation index of Ki67 was significantly decreased,which was significantly different from that of control group.Conclusion:1 A retrospective study of clinical cases showed that PIAS3 expression in gastric cancer was negatively correlated with local tumor invasion and lymphatic metastasis.The expression level of PIAS3 in gastric carcinoma was positively correlated with the overall survival of gastric cancer.It suggested that PIAS3 was expected to be a potential biomarker for the prognosis of gastric cancer.2 In vitro and in vivo experiments showed that PIAS3 expression was decreased in gastric cancer cells and negatively correlated with STAT3expression.Overexpression of PIAS3 could downregulate STAT3 activity and significantly inhibit the proliferation and metastasis of gastric cancer cells,suggesting that PIAS3 could inhibit STAT3 Expression of gastric cancer cell proliferation and metastasis,PIAS3 is a tumor suppressor gene in gastric cancer. |
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