| The main challenge in the identification and characterization of miRNA function is the gain of a comprehensive understanding of their effects on the expression of the targeted mRNAs and the resulting outcome on relevant cellular pathways and phenotypes. In order to achieve this purpose, we need to know the functional targets of each microRNA in vivo. Both of the experimental and bioinformatic data demonstrate that each microRNA could regulate hundreds even thousands of target genes. However, the current bioinformatic methods could not accurately predict the authentic targets of a microRNA. Therefore, in order to deeply understand the biologic function of a microRNA, using effective method is the key.In the first part of this thesis, we used the miRACE (miRNP isolation and miPrime RACE) method which was developed by our lab to systematically identify the conservative and non-conservative targets of miR-16-1 in tumor cells, and we have obtained total 330 potential targets of miR-16-1. Then we analyzed the target site and did GoTerm analysis, which uncovered the noval mechanisms and biological functions of miR-16-1. Progression of a cell through or its arrest in a specific cell cycle phase requires the integration, processing and initiation of a variety of signal transduction pathways. Critical for the function of the cell cycle machinery are the activities of cyclin dependent kinases (Cdks) and their activating subunits, the cyclins. Cyclin E1 (CCNE1), an essential cyclin activating Cdk2, regulates the G1-S phase transition of the mammalian cell division cycle. In normal dividing cells, its timing expression plays a direct role in the initiation of DNA replication, the control of histone biosynthesis, and the centrosome cycle. In numerous human tumors, however, CCNE1 expression is frequently dysregulated, even overexpressed, while the mechanism leading to its dysregulation remains incompletely defined. In the second part of this thesis, we showed that CCNE1 expression was subject to post-transcriptional regulation by a microRNA miR-16-1, a newly appreciated class of post-transcriptional regulatory elements.A bioinformatics search revealed two evolutionary conserved target sites for miR-16-1 within CCNE1-3’-UTR at nt229-254 and nt459-492. Ectopic expression of miR-16-1 dramatically reduced endogenous protein level of CCNE1 as well as its mRNA level. Furthermore, transfection with miR-16-1 significantly suppressed the expression of a luciferase reporter carrying the 3’UTR region of CCNE1, whereas transfection with anti-miR-16-1 obviously increased the activity of this construct. This was almost abolished when constructs mutated at the putative miR-16-1 target sites were used instead. This thesis also found that enforced expression of miR-16-1 induced a significant G1 cell cycle arrest and anti-miR-16-1 reversed this phenotype. Further investigations demonstrated that silencing of CCNE1 partially phenocopied the effect of miR-16-1 and substantially rescued anti-miR-16-1-induced cellular phenotype. In aggregate, this thesis suggests that miR-16-1 plays a key role in regulating cellular processes in human cancers and implicate the potential application of miR-16-1 in cancer therapy. |
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