MiR-200c/Zeb1Negative Feedback Loop-a Regulator Of Epithelial-Mesenchymal Transition And Tumorigenicity In Intraocular Tumors

AimsTo investigate the regulation effect of microRNA-200c/Zeb1feedback loop on Epithelial-Mesenchymal transition (EMT), tumorigenic capacity and motility of the intraocular malignant tumor cells.Metholds1.①.Build up miR-200c over-expression and inhibited cell models in intraocular tumors. Hsa-miR-200c mimics or hsa-miR-200c inhibitor was transfected in combibation with LipofectamineTM2000to human uveal melanoma cell line OCM-1, human retinoblastoma cell lines WERI-RB1and Y79. The transfection efficacy were checked by percentage of FAM-positive cells under fluorescent microscope and relative quantities of miR-200c expression.②Indentify the correlation between miR-200c and Zeb1. Before and after transfection,the expression of its target gene Zeb1were detected by real time RT-PCR, immunoblotting and Immunofluorescence in RNA and protein level respectively.③To detect the transition between epithelial and mesenchymal phenotype induced by miR-200c intervention in intraocular tumor cell lines. The RT-PCR, Western-blot and Immunofluorescence were applied to detect the expression of epithelial-specific gene E-cadherin, mesenchymal-specific genes Vimentin and N-cadherin in different levels.④To inspect the mobility changes of the tumor cells after the EMT or MET process. Transwell chamber was used to test the in vitro migration force of the three intraocular tumor cells under different intervention.2.①To determine whether the expression of stem cell-specific gene Bmi-1was also under control of miR-200c, real time RT-PCR and immunoblotting were practiced to quantify the Bmi-1in RNA and protein level respectively.②We test the in vitro tumorigenicity of the OCM-1with plate clone exam, to compare the clone formation effeciency of the cells under different intervention.3.①For the requirement of the further in vivo examination, we transfect OCM-1cells with lentivirus particles containing Zeb1cDNA and selected the stable over-expression OCM-1cells by puromycin-resistant gene.②In order to evluated the function of Zeb1gene on epithelial-mesenchymal transition, tumorigenicity, we quantified expression of Zeb1、miR-200c、E-cadherin、Vim entin、 N-cadherin and Bmi-1by real-time RT-PCR in RNA level and Western-blot in protein level.③The phenotype of the stable transfected cells were investigated by microscopy.Results1.①miR-200c over-expressed and inhibited cell models were successfully build up in OCM-1, WERI-RB1and Y79cell lines. The relative quantification of miR-200c in the mimics transfection groups were significantly up-regulated when compared with the control groups (p<0.05). On the other hand, the inhibitor group showed significant decrease of miR-200c(p<0.05).②Transient transfection of the miR-200c mimics into the caused a marked repression of Zeb1in both mRNA and protein levels, while the miR-200c inhibitor improve the expression of Zeb1. The relative quantification of the mRNA changes were statistical significant (p<0.05).③The transfection of miR-200c resulted to a promotion of E-cadherin expression, the differences of mRNA relative quantification were statistically significant(p<0.05). At the same time, the express of Vimentin and N-cadherin were suppressed by the overexpression of miR-200c. While the transfection of miR-200c inhibitor result in a complete reverse regulation when compared with mimics.④The trans-membrane mobility and tumor of OCM-1, WERI-RB1and Y79cells can be inhibited by miR-200c. The quantification of trans-membrane cells under intervention of miR-200c mimics were significat less than the control, while the inhibitor transfection group had more migrated cell than the control. 2.①Over-expression of miR-200c can inhibite the expression of stem-cell factor Bmi-1in both mRNA and protein level when investigatd with RT-PCR and Western-blot. The differences of mRNA between miR-200c mimics groups and controls were statistically significant. Transfection of miR-200c inhibitor induced a completed reverse result.②miR-200c could down-regulate the tumorigenic ability of OCM-1cells. The sphere-forming rates of the control, mimics and inhibitor groups were31.6%,22.8%and57.9%respectively.③over-expression of miR-200c can induce some OCM-1cells live in an epithelial mode and phenotype, while the miR-200c inhibitor can make the tumor cells transit to a mesenthymal mode and fusiformal phenotype.3.①The stable Zeb1over-expressed COM-1cells were aquired.②The relative quantification of Zeb1mRNA had5.49-fold increase compare with those of controls.③The over-expression of Zeb1can significantly repress the expression of miR-200c in OCM-1cells. After the stable transfection,the relative quantification of miR-200c expressed67%reduction.④Transfection also induced a depression of E-cadherin and increases of Vimentin, N-cadherin and Bmi-1in Zebl over-expressed OCM-1cells. The mRNA differences between transfeced group and control were all statistical significant (p<0.05).⑤The selected stable Zeb1over-expressed OCM-1cells showed significant mesenchymal phenotype changes. In addition to the mesenchymal growth pattern and fusiformal morphology which had been generally reported, we also detected a lot of dentric cells and significant magnified nuclei. The mechinism of the phenotype changes and the function of the stable cell line require further in vitro and in vivo investigation.ConclusionmiR-200c/Zeb1feedback loop play a very important regulation effect on EMT, tumorigenicity and mobility of intraocular tumor cell lines. Dramatic morphological changes observed in the stable OCM-1cells with over-expressing Zeb1gene might involve some unknown mechanisms more than EMT.