The Research Of Effects Of MicorRNA-200b/c On Invasion And Cancer Stem Cell Properties Of Cholangiocarcinoma

Part I The Screening and Validation of differential microRNA on cholangiocarcinomaObjective To identify miRNAs differentially expressed in cholangiocarcinoma and corresponding normal bile duct tissues. To validate the results and define the research objective for future experiments.Methods To identify the expression of miRNAs in three cholangiocarcinoma and corresponding normal bile duct tissues by an Agilent miRNA Microarray. The differential microRNA and target genes were analyzed by biological processes and the molecular function using information available from the Gene Ontology (GO) and pathway database.The results of microarray were validated by RT-qPCR and Northern blot.And the results were used for clinlical analyses.Results Fifteen miRNAs were overexpressed and six miRNAs were underexpressed in the cholangiocarcinoma tumor group compared with that in the controls. MiRNA levels in the cholangiocarcinoma group were2.25-to42222-fold overexpressed, and were6.39-to4032-fold under-expressed in the controls. there were78up-regulated GO terms and82 down-regulated terms.And we performed pathway analyses and found20up-regulated pathways and30down-regulated pathways in relation to the dysregulated genes. Three miRNAs, miR-200b, miR-200c and miR-429, were selected to validate the microarray results by RT-qPCR and Northern blot. Data analysis showed that miR-200b, miR-200c and miR-429were underexpressed in the cholangiocarcinoma group compared with that in the controls (P=0.038and P=0.019, respectively).The expression levels of miR-200b/c associated with the degree of pathological differentiation (P<0.001).Conclusion The miR-200b/c is validated as differential microRNA of cholangiocarcinoma and chose for future experiment. Part Ⅱ The research of effects of miR-200b/c on invasion, metastasis and chemoresistance of cholangiocarcinomaObjective To identify the effects of miR-200b/c on invasion,metastasis and5-fluorouracil chemoresistance of cholangiocarcinoma cells.Methods Transfection of miR-200b/c mimics and antagomir into TFK-1cells resulted in a substantial increase and decrease of miR-200b/c expression, respectively.And the transfdcted cells were used for Transwell invasion, wound-healing assay and5-fluorouracil drug resistance array. To transfect TFK-1cells with a lentivirus-based expression vector to overexpress and down-regulate the expression levels of miR-200b/c, respectively. Next, we established a liver metastasis model of cholangiocarcinoma using these transfected cells.Results The invasion assay showed that overexpression of miR-200b/c resulted in a reduction of the invasion rate of TFK-1cells compared with that of the control (5.04±0.11-and3.12±0.03-fold, respectively, P<0.001). TFK-1cells transfected with miR-200b/c mimics slowly closed the wound gaps compared with that of the control (2.13±0.24-and1.92±0.03-fold, respectively, P<0.001). Knockdown of miR-200b/c in TFK-1cells resulted in significant closing of the gap in the wound healing assay (0.69±0.01-and0.65±0.03-fold, respectively, P<0.001) and a reduction of cell invasion compared with that of the antagomir-NC (1.96±0.10-and3.07±0.19-fold, respectively, P<0.001). IC50values of the miR-200b/c mimics+5-fluorouracil group were significantly lower than that of the negative control group.Up-regulation of miR-200b/c inhibited the distant metastasis of TFK-1cells and knockdown of miR-200b/c promoted this metastatic process.Conclusion MiR-200b/c is an important participant in the regulation of migration and invasion potentials and enhances the cytotoxic effect of5-fluorouracil on cholangiocarcinoma cells. Part Ⅲ The research of effects of miR-200b/c on cancer stem cell properties of cholangiocarcinomaObjective To identify the effects of miR-200b/c on cancer stem cell properties of cholangiocarcinoma.Methods To investigate the effects of miR-200b/c on tumor formation, we subcutaneously injected TFK-1cells transfected with control, miR-200b/c overexpression and knockdown vectors, respectively, into the right armpit of BALB/c nude mice. Then the transfected cells were cultured in suspension under serum-free conditions to generate spheres for self-renewal and differentiation assay.To detect the CD133proportion using flow cytometry.Results MiR-200b/c-overexpressing cells formed tumors much slower than those in the negative control group. In addition, miR-200b/c-knockdown cells showed the opposite trend (P<0.001). The weight of mice also had statistical significance from week4(P<0.01). miR-200b/c-overexpressing cells formed less spheres than those in the NC group (P<0.001). The three passages of miR-200b/c-knockdown cells formed more spheres than those in the NC group (P<0.001). MiR-200b/c-knockdown cells formed larger spheres than those of the controls (P<0.001), while miR-200b/c-overexpressing cells formed smaller spheres than those of the controls (P<0.001). The Expression levels of miR-200b/c decreased after formation of spheres and increased during differentiation. The proportion of CD133+cells among miR-200b/c-overexpressing cells was lower than that in the negative control (P<0.001). The proportion of CD133+cells among miR-200b/c-knockdown cells was high (P<0.001). The proportion of CD24+and CD44+cells was not related to miR-200b/c expression.Conclusion The capacity of TFK-1cells was associated with low miR-200b/c expression. MiR-200b/c-overexpressing spheres may have undergone the first step toward losing their self-renewal capacity. MiR-200b/c-knockdown spheres may have a greater capacity for sphere formation, indicating a higher self-renewal capacity. CD133as a potential CSC marker of cholangiocarcinoma can be inhibited by miR-200b/c. Part Ⅳ The research of target gene and mechanism of miR-200b/c on invasion, metastasis and cancer stem cell properties of cholangiocarcinomaObjective To identify the target gene and their downstream sites of miR-200b/c effecting invasion, metastasis and cancer stem cell properties of cholangiocarcinoma.Methods TargetScan analysis and luciferase reporter assay were used for target gene analysis of miR-200b/c. The mRNA expression of SUZ12and ROCK2were detected by Northern Blot. The protein expression of SUZ12and ROCK2were detected by Western Blot and Immunohistochemistry. The functional experiments were carried out as described before.Results Both SUZ12and ROCK2were overexpressed in cholangiocarcinoma tissues (nine of12and six of12tumor tissues showed increased SUZ12and ROCK2levels, respectively, compared with those in matched normal bile duct tissues). Immunohistochemistry showed that the protein expression levels of SUZ12and ROCK2were higher in tumor tissues than those in normal bile duct tissues (P=0.018and P<0.001, respectively). There was little significant difference of mRNA expression levels of SUZ12/ROCK2between the cholangiocarcinoma and normal bile duct groups. The SUZ12/ROCK2wild-type sequence, but not a mutant sequence, was transfected into TFK-1cell, we found that ectopic expression of miR-200b/c resulted in a decrease of luciferase activity. Silencing of ROCK2and E-cadherin decreased the number of invasive cells compared with that of the controls. In addition, silencing of both ROCK2and E-cadherin expression resulted in the lowest number of invasive cells. The SUZ12shRNA group formed smaller spheres than those in the negative control. The spheres of the control group increased in size and cell number more quickly than those of the SUZ12shRNA group.Conclusion SUZ12and ROCK2are direct targets of miR-200b/c. The effect of miR-200b/c on the invasion capacities of cholangiocarcinoma cells was mediated not only via the Zebl/2-E-cadherin axis, but also the Rho/ROCK2pathway. SUZ12is an important gene related to cholangiocarcinoma CSC-like properties, and is regulated by miR-200b/c.