Research On The Expression And Clinical Significance Of GOLPH3 And NLRP3 In Gallbladder Carcinoma And The Mechanism Of Their Effects On The Proliferation In Gallbladder Carcinoma Cells

Part one: The expression of GOLPH3 and NLRP3 in gallbladder carcinoma and their clinical significance Objective To investigate the expression and clinical significance of Golgi phosphoprotein 3（GOLPH3）and NOD-like receptor protein 3（NLRP3）in human normal gallbladder tissue,gallbladder precancerous lesion tissue and gallbladder carcinoma（GBC）tissues.Methods Surgical specimens and clinical data of 63 patients with GBC who had undergone radical cholecystectomy in the 904 th Hospital of Joint Logistic Support Force of PLA from January 2014 to January 2019 were collected.In the GBC group,there were 21 males and 42 females,with an average age of 62.5 years.For 30 patients with mild to moderate atypical hyperplasia of gallbladder during the same period were sampled and included in the precancerous lesion group,including 9 males and 21 females,with an average age of 62.4 years.Normal gallbladder specimens from 20 patients who underwent surgical resection due to liver trauma or giant hepatic hemangioma were collected and included in the normal group,including 7 males and13 females,with an average age of 61.9 years.The expressions of GOLPH3,NLRP3,Ki-67 were detected by immunohistochemistry.The positive rate of them was counted,and the correlation between GOLPH3,NLRP3 and Ki-67 was analyzed.Log-rank test and Cox regression analysis of the GOLPH3 and NLRP3 expression and survival prognosis of GBC patients.Results 1.The positive rates of GOLPH3,NLRP3 and Ki-67 were 71.4%（45/63） and 68.3%（43/63）in the GBC group and 43.3%（13/30）and 36.7%（11/30）in the precancerous lesion group,and 15.0%（3/20）,5.0%（1/20）in the normal group,respectively.The expression of them in the tumor group,precancerous lesion group and normal group decreased in order.2.In GBC tissues,the positive rate of GOLPH3 expression and NLRP3 expression was positively correlated with the positive rate of Ki-67 expression（r =0.972 and r =0.969,both P < 0.05）.3.The positive rate of GOLPH3 expression and NLRP3 expression was positively correlated（r=0.972,P<0.05）,and the kappa test showed that the positive expression of GOLPH3 and NLRP3 in patients was positively correlated（Kappa=0.774,P<0.05）.4.Compared with negative patients,patients with GOLPH3 positive expression had differences in the aspects of tumor location（neck vs.bottom and body）,depth of infiltration,lymph node metastasis,differentiation degree,growth pattern,TNM staging,preoperative CRP（P<0.05）.Patients with NLRP3 positive expression had differences in the aspects of tumor location（neck vs.bottom body）,depth of infiltration,lymph node metastasis,differentiation degree,growth pattern,TNM staging,preoperative bilirubin levels,CRP and CA199 levels（P<0.05）.5.The follow-up period was 1 to 68 months,and the median follow-up time was 23.6 months.Univariate analysis showed that postoperative survival of patients with GBC was related to tumor location,vascular invasion,lymph node metastasis,tumor differentiation,TNM staging,R0 resection,postoperative chemotherapy,preoperative carbohydrate antigen 19-9,GOLPH3 and NLRP3 protein expression（All P<0.05）;Multivariate analysis showed that positive GOLPH3（HR=4.891,95%CI: 1.776-13.470）and positive NLRP3（HR=3.006,95%CI:1.273-7.099）were independent risk factors for postoperative survival of patients with GBC.Conclusion 1.GOLPH3 and NLRP3 are highly expressed in GBC tissues and positively correlated.2.GOLPH3 and NLRP3 are positively correlated with the expression of Ki-67 in GBC tissues.3.The positive expression of GOLPH3 and NLRP3 is independent risk factor for postoperative survival in patients with GBC.Part two: Golgi phosphoprotein 3 promotes the proliferation of gallbladder carcinoma cells by regulating NLRP3Objective To explore the mechanisms of GOLPH3 and NLRP3 promoting the proliferation of GBC cells,and provide new potential molecular targets for the treatment of GBC.Methods 1.In GBC-SD cells,GOLPH3 SiRNA and overexpression plasmid were transfected via Lipofectamine^TM2000.The interference and overexpression effects of it were detected by WB,and cell proliferation was detected by CCK-8 and EDU.2.The si RNA and overexpression plasmid of GOLPH3 were transfected into GBC-SD cells,and the expression level of NLRP3 and Caspase-1/P10 protein was detected by WB.3.The NLRP3 si RNA was transfected in GBC-SD cells,and the level of GOLPH3 and Caspase-1/P10 protein was detected by WB.4.Simultaneously overexpress GOLPH3 and down-regulate NLRP3 in GBC-SD,and detect the proliferation of GBC cells through CCK-8 and EDU experiments in the co-transfection experimental system.Results 1.Down-regulation of GOLPH3 inhibited the proliferation of GBC-SD cells,and overexpression of GOLPH3 promoted the proliferation of GBC cells.2.After down-regulating and over-expressing the expression of GOLPH3,the level of NLRP3 and Caspase-1 P10 protein decreased and increased respectively.3.After down-regulating NLRP3,the level of Caspase-1 P10 protein decreased,while the expression of GOLPH3 remained unchanged.4.Simultaneously over-expression of GOLPH3 and down-regulation of NLRP3 protein expression,the level of Caspase-1 P10 and the proliferation of gallbladder cancer cells GBC-SD have no significant changes compared with control group.Conclusion In GBC-SD cells,GOLPH3 promotes tumor cell proliferation via regulating the expression level of NLRP3 and activating Caspase-1 protein.