Development of a Novel MicroRNA Screening System by Identifying the Candidate MicroRNAs that Target Oct4 Öffentlichkeit
Nguyen, Phan (2012)
Abstract
MicroRNAs (miRNAs) are posttranscriptional modulators of gene expression that act by directly targeting its 3' untranslated region (3'UTR) and play an important role in many developmental processes. Aberrant regulation and altered expression of specific miRNA genes contribute to the initiation and progression of cancer stem cells (CSC). To date, more than 1000 human miRNAs have been documented, but the functions of most of them remain elusive. The main purpose of this study is to set-up an efficient miRNA screening system of a total of 931 human miRNAs by combining standard transfection protocols with high throughput screening (HTS) and high content analysis (HCA) equipments from the Emory Chemical Biology Discovery Center (ECBDC). Oct4 was chosen as the initial target gene since it is an essential transcription factor in embryonic stem cell (ESC), as well as cancer stem cell (CSC), development and it is a small gene with a short 3'UTR, which allowed for easy initial set-up and modifications of this novel screening system. The system uses fluorescence signals, which correlate to the targeting effects of miRNAs on target genes, to narrow down the large miRNA library to facilitate focused studies with a more manageable miRNA sample size. The initial screening identified miR-4310 and miR-1253 as candidates that target Oct4-3'UTR. Western blotting results verified the target relationship of Oct4 and the miRNAs and further confirmations of the findings by luciferase assays and miRNA dose response curve to pDsRed2-Oct4-3'UTR will be followed. The identification of these miRNA candidates against Oct4 from a library of 931 miRNAs revealed that the new screening approach is promising and efficient, although improvements are still necessary to strengthen the system. Nonetheless, with the establishment of this novel and automatic miRNA screening system, the 3'UTR of any desired gene can be screened for its miRNA regulators within a reasonable time frame, while also reducing the extensive labor, human error, and supply cost associated with manual screenings.
Table of Contents
Abstract...1
Introduction...2
Scheme 1. miRNA target screening
design...6
Scheme 2. Experimental design...8
Materials and Methods...13
Results...16
Figure 1. miRNA expression plasmid...17
Figure 2. 293FT cells confluencies of initial seedings at different
densities...18
Figure 3. pDsRed2-Oct4-3'UTR plasmid...19
Figure 4. Dose response transfections of pDsRed2- 3'UTR of p53 and
Oct4...22
Figure 5. p53-3'UTR positive control confirmation...24
Figure 6. Plate 1 of miRNA and pDsRed2-Oct4-3'UTR
co-transfections...26
Figure 7. 931 miRNAs' DsRed2 % iInhibition rankings...28
Table 1. miRNA with ≥ 50% pDsRed2 Inhibition...29
Figure 8. pCMV-HA-Oct4-3'UTR plasmid and western blot
verifications...32
Discussion...33
References...39
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