The Role Of Eukaryotic Translation Initiation Factor 4E In The Progression Of Gallbladder Cancer

BackgroundGallbladder carcinoma is one of the most common and invasive malignancies of the biliary tract system.Gallbladder cancer is the seventh most common cancer of the digestive system in China,accounting for about 1%of all cancer cases,with a incidence of 3.82 per 100,000 people.Because of the lack of specific symptoms,lack of effective treatment and prognostic indicators,the prognosis of gallbladder cancer is still not satisfactory.Epidemiological statistics show that the overall survival of gallbladder cancer is about 6 months,and the 5-year survival rate is less than 10%.Therefore,it is of great theoretical and clinical significance to study the pathogenesis of gallbladder cancer,which is helpful to provide theoretical basis,new treatment strategies and new means for the prevention and treatment of gallbladder cancer.Morphology and DNA sequencing studies have further confirmed the proposed adenoma-gallbladder carcinoma hypothesis,which describes the disease progression of gallbladder carcinoma.Recent studies suggest that the adenoma-gallbladder carcinoma hypothesis plays a more important role in tumor formation in young patients with gallbladder carcinoma than the metaplasia-dysplasia-gallbladder carcinoma hypothesis.Mammalian rapamycin receptor(mTOR)signaling pathway and mitogen-activated extracellular signal-regulated kinase(MEK)signaling pathway play important roles in the progression of gallbladder cancer.Eukaryotic translation initiation factor 4F(eIF4F),as a key regulatory target in the initiation stage of translation,is the common action point of mTOR pathway and MAPK pathway signal transmission.Eukaryotic translation initiation factor 4E(eIF4E)is a cap-binding protein that specifically recognizes the 5’cap structure of mRNA.eIF4E,together with eukaryotic translation initiation factor 4A(eIF4A)and eukaryotic translation initiation factor 4G(eIF4G),constitutes eIF4F and participates in eukaryotic translation initiation.eIF4E level affects the translation of a series of mRNAs encoding proliferation and promoting tumorgenesis proteins on some degrees.Collectively known as eIF4E-sensitive mRNAs,these mRNAs include cyclin,ornithine decarboxylase,vascular endothelial growth factor(VEGF),and phosphate ribose pyrophosphate synthase-2.Recently,the role of eIF4E in tumors has been of great interest to researchers.eIF4E expression was increased in tumor tissues.For example,for facial squamous cell carcinoma,myc-mediated transcriptional activation gene amplification leads to the overexpression of eIF4E.eIF4E overexpression at the edge of tumor indicates that recurrence is likely to be large and is negatively correlated with survival.In this study,we tested the expression level of eIF4E in the non-diseased gallbladder tissue,gallbladder adenoma and gallbladder cancer,analyzed the differences of eIF4E expression in three groups of tissue samples and the correlation between eIF4E expression level and clinicopathological data and prognosis of gallbladder cancer patients statistically.Material and methods1.Patients and samples:The present study was approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University(Hefei,China),and all patients provided written informed consent.A total of 76 GBC specimens were obtained from GBC patients who underwent radical cholecystectomy at the General Surgery Department between November of 2011 and November of 2016.All patients who were enrolled in the study did not receive any prior treatment.The control group included 58normal gallbladder tissues,of which 21 specimens were obtained from patients who were diagnosed with hepatic hemangioma and underwent hepatectomy combined with cholecystectomy,and the other 37 gallbladder adenoma tissues obtained from patients with gallbladder adenoma who underwent laparoscopic cholecystectomy(TableⅠ.).Anatomic and histologic grades were established according to the criteria by American Joint Committee on Cancer(AJCC)tumor node metastasis(TNM)Staging for Gallbladder Cancer.2.Immunohistochemistry(SABC)method was used to determine the expression level of eIF4E in each group of tissue samples.Under the Nikon Eclipse 8ic microscope,5400-fold discontinuous fields were randomly selected for each section,and the Nikon Digital camera was used to record and save the images to be measured.The average optical density(OD)value of eIF4E staining in the images obtained by the Image Pro Plus 6.0 Image analysis system was taken as the measured value.3.Cell experiment:Gallbladder cells were infected by slow virus.Then,the changes of the phenotype of gallbladder cell function caused by knockdown of eIF4E was evaluated by the cell proliferation experiment,colony forming test.the changes of the cell cycle caused by knockdown of eIF4E was detected by flow cytometry,the changes of the expression of cell cycle related proteins were analysised by using Western blotting.4.Xenograft model:A total of 5x10~6 NOZ cells expressing control shRNA or eIF4E shRNA were individually injected into the dorsal flanks of 4-week-old male athymic nude mice(Nanjing SLAC Laboratory Animal Co.Ltd.)(n=5 per group).The mice were randomly assigned to groups in the experiment(sh-eIF4E-1:5 nude mice,sh-eIF4E-2:5 nude mice).A total of 1 week after injection,tumors were measured per 4days with Vernier calipers and calculated using the following equation:volume=length x width~2 x 0.52.Then,4 weeks after injection,the mice were sacrificed,and the tumors were excised and weighed.During measurement of the weight of the tumors,the experimentalists were blinded to the information of tumor tissues.The excised tumors were homogenized,and proteins were extracted for western blot analysis,as aforementioned.Studies on animals were conducted on the basis of approval from the Animal Research Ethics Committee of the University of Science and Technology of China(approval no.,PXTG-MYD2017102611).5.Statistical analysis:SPSS 17.0 statistical software(SPSS Inc.,Chicago,IL,USA)was used for processing.Skewness distribution data were expressed as M(P25,P75).Kruskal-wallis H test was used to analyze the differences between the groups,and Mann-whitney U test was used to analyze the differences between the two groups.Normal data to?±s said,using the single factor analysis of variance.Bonferroni post-mortem correction was used.The classified data were analyzed by chi-square test.Cumulative survival rate was calculated by Kaplan-meier method,and log-rank test was used to compare the difference of survival rate.Cox regression was used for univariate and multivariate analysis.P<0.05 was considered statistically significant.Results1.The expression of eIF4E in non-diseased gallbladder group,gallbladder adenoma group and gallbladder carcinoma group showed a progressive increase.The expression of eIF4E in GBC was significantly higher than that in gallbladder adenoma,and the expression of eIF4E in normal gallbladder tissues was significantly lower than that in gallbladder adenoma.The differences between the three groups and between the two groups were statistically significant(P<0.05).2.The expression level of eIF4E increased gradually with the progression of gallbladder cancer,and was correlated with the overall survival of gallbladder cancer patients.The expression level of eIF4E protein in primary gallbladder cancer in T2-T4 stage was significantly higher than that in Tis-T1 stage,and the difference between the two groups was statistically significant(P<0.05).The expression level in the advanced gallbladder cancer subgroup(TNM II-IIIB)was significantly higher than that in the early gallbladder cancer subgroup(TNM 0-I),and the difference between the two groups was statistically significant(P<0.05).In addition,the expression level of eIF4E in the G3-G4(poor-undifferentiated)subgroup was higher than that in the G1-G2(high-moderately differentiated)subgroup,and the difference was statistically significant(P<0.05).Kaplan-meier analysis showed that the postoperative survival of the high-expression eIF4E subgroup of gallbladder cancer(OD≥0.3955)was lower than that of the low-expression subgroup,and the difference was statistically significant(P<0.05),the median postoperative survival of the eIF4E high-expression subgroup was 5 months,while the median postoperative survival of the eIF4E low-expression subgroup was 18 months.Postoperative survival of the advanced(TNM II-IIIB)gallbladder cancer subgroup was significantly shorter than that of the early(TNM 0-I)gallbladder cancer subgroup,with statistically significant differences(P<0.05).Multivariate analysis of Cox proportional hazard regression model showed that the up-regulated expression of eIF4E,TNM stage and histological grading were independent factors that affected the prognosis of patients with gallbladder cancer(P<0.05).3.Knockdown eIF4E inhibited proliferation and colony formation of GBC-SD and NOZ cells in vitro.The growth curve showed that the down-regulation of eIF4E expression significantly inhibited the proliferation of GBC-SD and NOZ cells(P<0.05).In line with the observation of cell growth curve,the colony formation ability of GBC-SD and NOZ cells infected with lentivirus containing eIF4E shRNA(sh-eIF4E group)was significantly decreased(P<0.05).In soft Agar,compared with sh-control group,the colony number of sh-eIF4E group was significantly reduced(P<0.05).After 96 h of lentivirus infection,the proportion of G0/G1 cells in sh-eIF4E group increased compared with that in the control group.(P<0.05)After infection with lentivirus containing eIF4E shRNA,the G2/M phase cell proportion of NOZ cells decreased(P<0.05).Western-blotting was used to further determine whether eIF4E regulates the expression level of cell cycle related proteins.The down-regulation of eIF4E resulted in decreased expression levels of cyclin D and cyclin E compared with the control group.At the same time,the expression of p27 was increased in GBC-SD and NOZ cells with low eIF4E expression level(P<0.05).4.The downregulation of eIF4E expression inhibited the xenograft tumor growth of NOZ gallbladder carcinoma cell line in vivo.To further explore the role of eIF4E in the development of GBC,we established a subcutaneous xenograft model in BALB/C nude mice using NOZ cells.Compared with sh-control,tumor growth was significantly inhibited in sh-eIF4E group,and tumor volume and weight in xenografts decreased in sh-eIF4E group.Further western blotting was used to detect the expression levels of p27,cyclin D1 and cyclin E1 in xenografts,and IHC was used to detect the expression levels of p27,cyclin D1,cyclin E1 and ki-67.Knockdown of eIF4E reduced the expression levels of cyclin D1,cyclin E1 and ki-67 in xenografted tumor tissues,while increased the expression levels of p27.ConclusionseIF4E is a potential independent risk factor for the prognosis of gallbladder cancer.It may play an important role in the progression of gallbladder cancer by affecting the proliferation of gallbladder cancer cells through cycle-related proteins.This study provides new insights into the pathogenesis of gallbladder cancer and new ideas for the treatment of gallbladder cancer.