The Study Of The Role And Expression Of RNA Helicase DDX5 In Intrahepatic Cholangiocarcinoma And Its Clinicopathological Significance

Part1 The biological role of DDX5 in ICC and the possible mechanisms ObjectiveThe objectives of the current study were to evaluate the biological function of DDX5 in the proliferation,metastasis,invasion and cell cycle of an ICC cell line(RBE)by downregulating the expression of DDX5 with a specific shRNA-DDX5,and to identify whether DDX5 has any connection with EMT process.MethodsWe constructed a lentiviral-mediated DDX5-shRNA and western blot were used to determine their expression levels of DDX5 protein.The group design consisted of a blank control group,a negative control group(NC group)and transfection groups(shRNA groups).Effects of DDX5 downregulation by shRNA in RBE cells were assessed by Alamar Blue assay(to evaluate the cell proliferation rate of RBE cells),flow cytometry analysis(to investigate the cell cycle distribution of RBE cells),wound healing assay(to validate the influence on migration ability),and transwell assay(to verify the influence on the invasion ability).Western Blot was employed to observe the related EMT marker(snail,EGFR,vimentin)protein levels in ICC cells of DDX5-shRNA group.ResultsWe successfully constructed 3 DDX5-shRNA and negative control shRNA.Of 3DDX5-shRNA,the first and the second were validated for the most efficient interference of DDX5 by western blot and was chosen for the following experiments.The results of Alamar Blue assay showed that the compared with the blank control group and negative control group,cell proliferation rate in DDX5-shRNA groups were significantly reduced at 48 h.Flow cytometry analysis illustrated that the percentage of cells in S phase increased and the corresponding proportion of G0/G1,G2/M phase decreased.Wound healing assay and transwell assay illustrated that the migration and invasion abilities of 2 DDX5-shRNA group cells were significantly inhibited(P < 0.05).Western blot results demonstrated that knockdown of DDX5 resulted in downregulation of snail,EGFR,and vimentin.ConclusionsIn vitro study,DDX5 downregulation could significantly inhibit the ICC cells proliferation,induce cell cycle arrest and suppress the ability of cell invasion and metastasis.The expression level of Snail,EGFR,and vimentin were downregulated,indicating that DDX5 is a key inducer of EMT.Part2 The significance of DDX5 in immunopathological diagnosis and clinical prognostic assessment of ICC ObjectiveThe main purposes of the present study were to investigate the value of DDX5 in ICC's differential diagnosis and its possible value as a potential therapeutic target,and to analyze correlations between the expression of DDX5,clinicopathologic parameters,and survival in ICC patients.MethodsTissue microarray(TMA)and IHC staining were used to analyze the association between DDX5 level,clinicopathological features and prognosis of DDX5 patients.The 323 ICC patients were divided into high and low expression groups according to the cut-off value calculated by X-tile statistical package.A total of 323paraffin-embedded ICC samples and the normal bile duct tissues were collected in our hospital from 2000 to 2006.The last follow-up date was May 2013.Survival was calculated by using log-rank test and Kaplan-Meier analysis.Stastic analysis was performed with SPSS 19.0 software.The level of statistical significance was set at P< 0.05.Immunohistochemistry(IHC)staining was used to evaluate the value of DDX5 in differential diagnosis with ICC,HCC(pseudoacinar type),BilIN-2,and metastatic adenocarcinoma.ResultsPositive IHC staining for DDX5 was observed primarily in the nuclear and cytoplasm.Of 323 ICC samples,52.5% were positive for DDX5.The expression of DDX5 was examined in ICC tissues and the normal bile duct tissues.Compared with the normal bile duct tissues,we found that DDX5 was significantly overexpressed in ICC tissues(P<0.05).Elevated DDX5 expression was correlated with sex,serum ALP(P<0.05),and shorter overall survival in ICC patients(P=0.011).Of 10 ICC samples,70% were positive;of 10 BilIN-2 tissues,40% were positive;of 10 HCC(pseudoacinar type)tissues,none of them were positive;of 10 metastatic adenocarcinoma,50% were positive for DDX5.ConclusionsCompared with the normal bile duct tissues,DDX5 was significantly upregulated in ICC tissues,and associated with clinicopathological features in ICC samples(sex,serum ALP).Patients with high DDX5 expression had poorer overall survival,whereas those with low DDX5 expression survived longer.Furthermore,DDX5 has value in differential diagnosis of ICC,BilIN-2,HCC(pseudoacinar type),and metastatic adenocarcinoma.