The Regulatory Loop Of MiR-101and The Proteomic Identification Of MiR-101Targets

Part1The Negative Feedback Loop between miR-101and EZH2Objective:To study the relationship between miR-101and EZH2in hepatoma cells. Methods:The relationship between miR-101and EZH2levels was investigated with gain-and loss-of-function assays. pre-miRNA-inducing experiments were used to confirmed functionality of miR-101/EZH2feedback loop and to determine which miR-101locus was involved in the loop. Quantitative genomic PCR was used to show whether miR-101locus status was correlated with pre-miR-101induction efficiency. The inhibitory effects of miR101on cancer cell migration and proliferation was evaluated by colony formation assays, migration/invasion assays, and wound healing assays. Results:Our data show that a reciprocal regulation feedback exists between EZH2and miR-101, and that this interaction is specific of miR-101-1and it is not affecting other miRNAs targeting EZH2such as miR-26. Moreover, miR-101expression inhibits cell proliferation and invasion, and this effect is reversed by EZH2overexpression. Conclusion:These findings establish a reciprocal negative feedback between EZH2and miR-101-1in HCC and highlight the coordinated role of miR-101and EZH2in hepatocarcinogenesis. Part2Two-Dimensional Electrophoresis-Based miRNA TargetDiscoveryObjective:To establish a2-D platform that is compatible with miRNA target discovery. Methods:A stable expression of miR-101in liver cancer cell lines was achieved by Lentiviral over-expression vectors. The total protein of miR-101overexpressing HepG2was separated with two-dimensional electrophoresis, with the empty vector cells as reference. Results:Over600protein spots were separated on a10%PAGE gel using24cm pH3to10nonlinear IPG strips. The reproducibility between the groups was satisfactory, with matching rate being more than99%. A total of20differential points was revealed after staining. Conclusion:2-D-based approach is compatible with miRNA target discovery. Part3LC-MS/MS-based miR-101targets identificationObjective:To identify miR-101downstream targets in hepatoma cells with LC-MS/MS. Methods:A total of15differential protein spots, which were cut manually, were lyophoilised and subjected to LC-MS/MS. Bioinformatics analysis was used to explore the possible connection of these differential proteins. Results:All of the15differential protein spots were identified successfully. Gene ontology reveal that these differential proteins were enriched in three groups, that is, RNA binding proteins, miR-200family targets and miR-101targets. Conclusion:Overexpression of mature miR-101resulted in a global change in the protein profile of HepG2cells and the majority of the identified proteins are associated with tumour formation.