Let-7c Controls Cholangiocarcinoma Growth But Promotes Tumor Cell Invasion And Growth At Extrahepatic Sites

Part Ⅰ Expression of let-7c is differentially regulated in both tumor tissues and sera of cholangiocarcinoma patientsObjective To identify miRNAs differential expression in cholangiocarcinoma and paratumor tissues and validate the results,then confirm the object of study for future experiment.Methods Deregulated miRNAs were identified in human bile duct cancer tissues by microarray analysis and confirmed by real-time PCR.Results microRNA let-7c expression was significantly downregulated in human cholangiocarcinoma tissues when compared to adjacent tissues of the same patient.In serum samples from the same patients let-7c levels were higher in patients with metastatic disease than in patients without metastasis.Conclusion microRNA let-7c is confirmed as differentially expressed miRNA in cholangiocarcinoma and selected for further study.Part Ⅱ Regulating the expression of let-7c can affect self-renewal ofcholangiocarcinoma cells in vitro and tumorigenic potential in vivo.Objective to clarify the self-renewal capacity and tumorigenic potential of cholangiocarcinoma are regulated by let-7c.Method Lentivirus with let-7c overexpressed,knockdown and control vector respectively transduced into TFK-1 cells to establish the stable cell line.Subcutaneously injected these transduced cells into the right armpit of BALB/c nude mice separately.The above mentioned stable TFK-1 cells were used to generate spheres in serum-free conditions for self-renewal and differentiation assay.Result Downregulated let-7c showed larger tumors at the injection site,while those injected with let-7c overexpressing cells showed smaller tumors,compared with the control group.Further,the cells,which overexpressed let-7c,formed tumors at a much slower rate than control group.The downregulated let-7c cells showed the opposite trend.There were significant differences in the weights of mice,from the fourth week amongst the three groups.Over three passages,the overexpressed let-7c cells formed fewer spheres than the cells in the NC group,whereas the cells with downregulated let-7c,formed more spheres than NC group.After six days under differentiation conditions,the expression level of let-7c decreased after sphere formation and increased during differentiationConclusion Regulation of let-7c in cholangiocarcinoma cells significantly affected their tumor-initiating capacity.Let-7c-overexpressing spheres may have undergone the first step toward losing their self-renewal capacity.In contrast,the spheres with low let-7c expression showed a greater ability for sphere formation,revealing a higher self-renewal capacity.Part Ⅲ Aberrant expression of let-7c inhibits migration and invasion ofcholangiocarcinoma cells in vitro but enhances metastasis in vivo.Objective To research the effect of let-7c on invasion and distant metastasis of cholangiocarcinomaMethod let-7c mimic and inhibitor were transfected into TFK-1 cells to increase or decrease let-7c expression,respectively.Then using these transfected cells to detect the invasion and migration of cholangiocarcinoma cells in vitro through transwell assay and wound healing assay.Using above mentioned stable transcriptional TFK-1 cells to establish distant metastasis mouse model.Result Invasion assay showed that the overexpressed Increased let-7c levels were associated with a lower invasion rates compared to scramble controls.TFK-1 cells,which were transfected with let-7c mimic,also showed decreased wound healing ability compared with the controls in the wound-healing assay;in contrast,downregulation of let-7c expression in TFK-1 cells facilitated the invasion of cholangiocarcinoma cells as well as significant gap closure in the wound-healing assay,compared with the scramble control group.Overexpression of let-7c promoted distant metastasis of TFK-1 cells and downregulation of let-7c lowered distant tumor foci.Conclusion Let-7c plays a differential role in regulating invasion and migration of cholangiocarcinoma,in vitro and in vivo.Part IV The research on target gene of let-7c and the mechanism in let-7c regulating the malignant biological behavior of cholangiocarcinomaObjective To clarify the target gene of let-7c and the mechanism of let-7c affected on invasion,distant metastasis and cancer stem cell like properties in cholangiocarcinomaMethod To predict target gene of let-7c with targetscan analysis and luciferase reporter assay.The expression of EZH2,DVL3 and beta-catenin in protein were detected by western blot and immunohistochemistry.The experiments of malignant biological behavior in cholangiocarcinoma were clarified before.Result Overexpressed let-7c in TFK-1 cells resulted in a reduction in EZH2,DVL3 andβ-catenin.In contrast,let-7c inhibition led to an increase in the expression of EZH2 and DVL3 and β-catenin.EZH2 and β-catenin,were overexpressed in cholangiocarcinoma tissues.We found that 8 of 11 tumor tissues and 9 of 12 tumor tissues overexpressed EZH2 and β-catenin,respectively,compared with the matched adjacent non-tumor tissues.Immunohistochemistry also showed that the protein expression levels of EZH2 andβ-catenin were higher in tumor tissues than in adjacent non-tumor tissues.The wild-type sequence of EZH2/DVL3 was transfected into TFK-1 cells,aberrant expression of let-7c lead to reduction in luciferase activity could be observed,but in mutant sequence of EZH2/DVL3,a decrease in luciferase activity was not observed.However,neither the wild-type sequence nor the mutated sequence of β-catenin showed a significant change in luciferase activity.Repressing EZH2 expression decreased the numbers of invading TFK-1 cells compared with the negative control.Decreasing expression of DVL3 lead to more distant metastasized foci compared with the negative control group.In addition,treated EZH2 with shRNA formed smaller spheres compared with NC group,furthermore,the sizes and number of spheres increased more quickly in NC group.Conclusion EZH2 and DVL3 are direct target genes of let-7c,while beta-catenin is not a direct target.However,let-7c may affect beta-catenin via the upstream gene DVL3.EZH2 and DVL/β-catenin are important genes involved in the malignant behavior of cholangiocarcinoma,and associated with migration,invasion,distant metastasis,sphere formation and tumor initiation,and they are regulated by let-7c.