Expression, Clinicopathological And Prognostic Significances, And Mechanisms Of A Novel Tumor Associated Gene, LAPTM4B, In Gallbladder Carcinoma

Background:Gallbladder carcinoma,which originated in epithelium of the gallbladder,carried concealed onset,rapid progression and dismal prognosis,thus seriously threatening the human health and life safety.In recent years,the increasing tendency of gallbladder carcinoma incidence in China further presented the necessity for thorough investigations on its pathogenesis and development.So far,domestic and abroad researchers have found that abnormal expression and mutation of many tumor associated genes,such as K-ras, P53,P27^(kip1),cyclin D1 andβ-catenin,existed in gallbladder carcinoma cells. Nevertheless,expression and its clinicopathological and prognostic significances,and relevant mechanisms of LAPTM4B(lysosome-associated protein transmembrane 4β),a novel tumor associated gene successfully cloned in hepatocellular carcinoma,remains unclear.Objective:The present study first,under the basis of previous relative literatures,explores the expression and clinical,pathological and prognostic significances,and further focusing on the effects and related molecular mechanisms of the gene on gallbladder carcinoma,in order to deepen the understanding on the pathogenesis and progression of gallbladder carcinoma and to probe into its new therapeutic targets.Methods:Part 1 Expression and clinicopathological and prognostic significances of LAPTM4B encoded proteins in gallbladder carcinomaWestern Blot:The expression of LAPTM4B encoded proteins is detected in paired cancerous and para-cancerous tissues,using polyclonal antibody LAPTM4B-EC2.Immunohistochemical staining:The expression of LAPTM4B-35 protein is detected by polyclonal antibody LAPTM4B-N_1-99,that specifically recognizes the protein,and is correlated with clinicopathological parameters of patients.Survival analysis:Survival rates are calculated by Kaplan-Meier method.Log-rank and Cox regression tests are adopted for uni-and multi-variate analyses.Part 2 Expression of LAPTM4B encoded proteins in gallbladder carcinoma cell line GBC-SDImmunocytochemical staining:The expression of LAPTM4B-35 protein is detected by polyclonal antibody LAPTM4B-N_1-99,that specifically recognizes the protein,in gallbladder carcinoma cell line GBC-SD.Western Blot:The expression of LAPTM4B encoded proteins in gallbladder carcinoma cell line GBC-SD is confirmed using polyclonal antibody LAPTM4B-EC2.Part 3 Mechanisms of LAPTM4B gene in gallbladder carcinoma cellsPlasmid amplification and identification:The plasmids pcDNA3-AE containing the complete open reading frame(ORF) of LAPTM4B and Mock(pcDNA3) are transformed into E.Coli DH5αand further amplified.The products are identified by digestion of endonucleases,BamH1 and EcoR1,and DNA sequencing.Plasmid transfection:Aforementioned plasmids are trainsiently transfected into gallbladder carcinoma cell line GBC-SD by liposome Lipofectamine^TM 2000.MTT assay:To detect proliferation of transfected and parent cells.Flow cytometry:To detect cell cycle distribution and apoptosis of transfected and parent cells.Transwell test:To detect migration and invasion of transfected and parent cells.Crossing river test:To detect migration of transfected and parent cells.Western Blot:To detect expression of related proteins in transfected and parent cells, using many kinds of specific antibody.Results:Part 1:LAPTM4B-35 protein is positively expressed in cancerous tissues of 2 patients out of 3 pairs of cancerous and para-cancerous tissues detected by Western Blot,whereas no any band is observable in para-cancerous tissues.Immunohistochemical staining shows that LAPTM4B-35 protein is positively expressed in cancerous tissues of 57(76%) patients with gallbladder carcinoma.Besides,its expression is closely correlated with histological type,lymph node involvement,staging and differentiation of the tumor,as well as patient prognosis.No positive staining is found in para-cancerous tissues.Part 2:Immunocytochemical staining reveals that LAPTM4B-35 protein is positively expressed in gallbladder carcinoma cell line GBC-SD.Western blot detection confirms this finding but simultaneously shows that its expression is a bit weak.Part 3:Digestion of two endonucleases and DNA sequencing indicate correct amplification.Cells transfected with pcDNA3-AE shows significantly accelerated proliferation and cell cycle progression,enhanced migration and invasion,and attenuated apoptosis induced by epirubicin.These phenotypes are accompanied by upregulated expression of c-myc,c-fos,c-jun,cyclin D1,cyclin E,MMP-2,MMP-9,uPA,Bcl-2 and Bcl-xL,and downregulated expression of P16,P27,Bax,Bid,cleaved caspase-3 and cleaved caspase-9.However,no similar effects are presented in cells transfected with the Mock plasmid and parent cells.Conclusions:LAPTM4B-35 protein is positively expressed in a majority of patients with gallbladder carcinoma and is of important clinicopathological and prognostic significances.LAPTM4B-35 protein is positively expressed in gallbladder carcinoma cell line GBC-SD.LAPTM4B gene promotes proliferation,migration and invasion,and inhibits apoptosis of gallbladder carcinoma cells.These effects are achieved via regulation of expression of multiple downstream genes.Therefore,LAPTM4B gene is of important implications in gallbladder carcinoma and may be a novel therapeutic target.