Effects Of ERK/AKT Signaling Pathway On MicroRNA-21 Inducing Epithelial-mesenchymal Transition Of Cholangiocarcinoma Cells

Introduction Cholangiocarcinoma(CCA) is a malignant tumor originated from the epithelial cells bile ducts. The rate of surgical resection is low and the recurrence rate is high. It is often associated with the early stage of tumor invasion and metastasis.Epithelial mesenchymal transition (EMT) is thought to be closely related to tumor invasion and metastasis, and EMT is confirmed to be regulated by multiple biological molecules and signaling pathways.MicroRNAs are a class of non-coding RNAs,which could regulate gene expression of target messenger RNAs negatively. The research of miRNA and its downstream regulatory genes has been the focus how microRNAs regulate EMT.Lots of studies have shown that extracellular signal regulated kinase (ERK1/2) and the 3-kinase/protein kinase B (PI3K-AKT) signaling pathway play an important role in the process of EMT in many kinds of tumors,but not in the studies about EMT of cholangiocarcinoma.Previous studies have found that miR-21 can promote the invasion and metastasis of bile duct cancer by regulating EMT, this study aims to explore the role of ERK and AKT signaling pathway in miR-21 inducing EMT in cholangiocarcinoma cell.Objective To observe the role of ERK and AKT signaling pathway in EMT induced by microRNA-21.Methods Scamble sequence, miR-21 mimes and miR-21 inhibitor were respectively constructed and transfected into QBC939 cells,which was cultivated in vitro.Cell group,21-NC group,21-M group and 21-I group were constructed. The expression of miR-21 and EMT related biomarkers(E-cadherin,N-cadherin,Vimentin) in each group were determined by real-time reverse transcription polymerase chain reaction(RT-qPCR). Then the expression of PTEN,ERK and AKT were determined by Western blotting. Then the expression of E-cadherin, N-cadherin, Vimentin were assessed by Western blotting and then Transwell assays were used to explore the invasion and migration cells when the ERK inhibitor(U0126) or AKT inhibitor (LY294002) was co-incubation with miR-21 in cholangiocarcinoma cells.Results No significantly difference was found between Cell and 21-NC group(P>0.05). the expression of miR-21,N-cadherin,Vimentin,p-ERK and p-AKT were upregulated while the expression of E-cadherin,PTEN were downregulated in 21-M group(P<0.05) when comparing with Cell goup,The expression of E-cadherin and PTEN were increased, while the expression of miR-21,N-cadherin,Vimentin.p-ERK and p-AKT were decreased in 21-1 group(P< 0.05).The expression of E-cadherin was upregulated,while N-cadherin and Vimentin were downregulated when the ERK inhibitor or AKT inhibitor was treated with miR-21.Meanwhile,the number of migration and invasion were decreased when ERK or AKT inhibitor was treated with miR-21 comparing with 21-M group.Conclusion:MicroRNA-21 can increase the expression of p-ERK、p-AKT、N-cadherin and Vimentin and decrease the expression of E-cadherin and PTEN.The ERK and AKT signaling pathway have been to play a role in miR-21-induced EMT,which has influence the ability of migration and invasion of cholangiocarcinoma.