Preliminary Study On The Mechanism Of EIF6 On The Intrahepatic Cholangiocarcinoma

Objective:Intrahepatic cholangiocarcinoma(Intrahepaticcholangiocarcinoma,ICC)is a primary malignant tumor that originating from bile duct epithelial cells.And its incidence is second only to hepatocellular carcinoma.Because of its lack of specific clinical manifestations,strong invasiveness and easy metastasis,the overall prognosis is poor.Therefore,it is significant to study the molecular mechanism for intrahepatic cholangiocarcinoma and actively search for new molecular targets to improve the prognosis of patients.Eukaryotic translation initiation factor 6(Eukaryotic translation initiation factor6,e IF6)belongs to the family of eukaryotic translation initiation factors,which can regulate the cellular translation process with other translation initiation factors.In recent years,many studies have found that e IF6 is differentially expressed in many solid tumors,suggesting that it may be involved in the development of multiple tumors.But so far,the role of e IF6 in intrahepatic cholangiocarcinoma is unclear.The purpose of this study is to detect the expression of e IF6 protein in intrahepatic cholangiocarcinoma and analyze its clinical significance.In addition,we hope to investigate the role of e IF6 in the formation of intrahepatic cholangiocarcinoma by silencing the expression of e IF6 in vitro and observing its effect on the biological behavior of human RBE cell lines,which can provide a new theoretical basis for finding possible therapeutic targets for intrahepatic cholangiocarcinoma.Methods:1 To detect the expression of e IF6 in ICC and the relationship with clinical and pathological characteristics.And to analyze the prognosis of patients with ICC.2 Transient transfection of e IF6-Si RNA was used to silence the expression of e IF6 in intrahepatic cholangiocarcinoma cell lines RBE,and the changes of cell biological behavior were observed.2.1 Si RNA e IF6-A/B was used to silence RBE cells and transfection efficiency was examined by Real-Time PCR and Western blot.2.2 Cell proliferation capacity assay and cell cycle effectsCCK-8 test and plate clone formation assay were used to detect the effect of silencing e IF6 on the proliferation of RBE cells in vitro.Flow cytometry was used to detect cell cycle.And western blot was to detect the expression of Cyclin B1 in RBE cells.2.3 Detection of migration and invasion of RBE cellsWound-healing test was used to detect the effect of silencing e IF6 on the migration of RBE cells.And transwell assay was used to detect the effect of silencing e IF6 on the invasive ability of RBE cells.2.4 Apoptosis related experimentsFlow cytometry was used to detect the effect of silencing e IF6 on the apoptosis of RBE cells.And western blot was used to detect the expression of apoptosis related proteins include BCL-2、BAX、Caspase-3、Caspase-9 in RBE cells.Results:1 The expression of e IF6 in intrahepatic cholangiocarcinoma tissues and its correlation with clinicopathological features1.1 Immunohistochemical results showed that the expression of e IF6 protein in ICC was higher than that in paracancerous tissues(P<0.05),which confirmed that the expression of e IF6 protein was up-regulated in ICC.Its expression was correlated with tumor differentiation and TNM stage(P<0.05),but not with sex,age,tumor size,tumor number,vascular invasion,capsule invasion and nerve invasion(P > 0.05).1.2 The survival of patients with high e IF6 expression was lower than that of patients with low expression.COX univariate and multivariate analysis showed that tumor differentiation and TNM stage were independent prognostic factors.2 Effect of silencing e IF6 on the biological behavior of RBE cells2.1 The results of real time PCR and Western blot showed that the relative expression of e IF6 gene m RNA and e IF6 protein in si RNA e IF6-A/B cells groups were significantly lower compare to control group(P < 0.001).2.2 Detection of cell proliferation and cell cycleThe results of CCK8 experiment showed that the OD values of the silent groups were lower than that of control group at four time periods(24h,48 h,72h,96h),suggesting that the proliferation ability of silent group was lower than control group(P < 0.05).The results of the plate clone formation assay showed that the number of colony formation of RBE cells in the silent group was significantly lower than that in the control group(P < 0.001).The results of flow cytometry showed that the number of cells in G1 phase was reduced in the silent group,while the number of cells in S phase and G2 phases was increased(P < 0.001).Meanwhile,Western blot showed that the expression of Cyclin B1 in silent group was lower than that of control group(P<0.001).2.3 Detection of cell migration and invasionThe results of Wound-healing test showed that the migration ability at 24 hours and 48 hours of RBE cells in the silent group was lower than that in the control group(P < 0.05).The results of transwell assay showed that the invasive ability of RBE cells in the silent group was lower than that in the control group(P < 0.001).2.4 Apoptosis related experiments resultsThe results of apoptosis detected by flow cytometry showed that the number of apoptosis of RBE cells increased after silencing the expression of e IF6(P <0.001).The results of Western blot suggested that the expression of Bcl-2,Caspase-3 and Caspase-9 protein in RBE cells were lower in silent group.while the expressions level of Bax was higher in silent group(P < 0.001).Conclusions:1 The expression of e IF6 is upregulated in ICC,and the expression of e IF6 is correlated with tumor differentiation and TNM stage.The survival of ICC patients with high e IF6 expression was lower than that of patients with low expression.And tumor differentiation and TNM stage were independent prognostic factors of ICC.2 Silencing e IF6 can inhibit the proliferation,migration,and invasion ability of RBE cell,block cell cycle in G2 / M phase and promote apoptosis.