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Introduction
MicroRNAs are ~22 nt endogenous RNAs that function to fine-tune the
expression of a target gene. Most mammalian miRNAs are found to decrease
target mRNA levels resulting in decreased protein production [1]. It has
been theorized that most mammalian genes are targets of miRNAs [2].
Currently, the only method to screen which miRNAs may target a gene of
interest is through prediction algorithms. Different prediction algorithms
use slightly different rules which can lead to substantially different
results. Even after using prediction algorithms to determine which miRNAs
target a gene of interest, these miRNAs must be tested experimentally to
verify that they are real interactions. For a gene that may have tens or
hundreds of predicted miRNAs, this can be an expensive and daunting task.
In addition, prediction algorithms may miss true miRNA/target RNA
interactions due to the rules that they are restricted to. miRNA
Transcriptome PCR Array provides comprehensive experimental methods to
screen for miRNAs that target a gene of interest.
To demonstrate the power of miRNA-FIND in discovering novel miRNA/mRNA
interactions, we screened for miRNAs that target ZEB2. ZEB2 is a
transcription factor that has been implicated in the epithelial-mesenchymal
transition (EMT).
Material and Methods
In this study we used "Cancer miRNA SureFind Transcriptome PCR
Array" to identify potential miRNAs that target ZEB2. SYBR Green qPCR
assay was performed to quantify the expression of ZEB2 (gene of interest)
and GAPDH (house-keeping control). Fold change in ZEB2 gene expression as a
result of each specific miRNA mimic treatment relative to negative mimic
control were calculated and normalized to GAPDH. Fold changes were
converted to log2 and subjected to MAD analysis for positive hits
selection.
Figure 1: miRNAs involved in regulation of ZEB2 gene
expression. Cancer miRNA SureFind Transcriptome PCR Array was used to run SYBR
Green-based qPCR assays for ZEB2 and GAPDH. ZEB2 gene expression level is
expressed as Log2 fold change based on Ct calculation using GAPDH as
house-keeping gene and non-target miRNA mimic treated sample well (VTC) as
negative control.
Results
Our results show that miR-200c, let-7g, miR-122, miR-142-5p, and miR-29b
negatively regulate ZEB2 gene expression (Fig. 1). Also, miR-34c-5p caused an
increase in expression, possibly identifying it as a positive regulator of ZEB2
mRNA expression (Fig. 1). Interestingly, miR-200c has been shown to inhibit EMT
through direct targeting of ZEB2 [3]. While miR-200c has been previously
identified to target ZEB2, let-7g, miR-122, miR-142-5p, miR-29b and miR-34c-5p
are novel regulators that are not identified by any prediction algorithm (data
not included).
Conclusions
miRNA Transcriptome PCR Array identified six miRNAs that regulate ZEB2
expression and five of these (let-7g, miR-122, miR-142-5p, miR-29b and
miR-34c-5p) were not predicted by bioinformatics (data not included).
References
1. Guo H, Ingolia NT, Weissman JS, Bartel DP. Mammalian microRNAs predominantly
act to decrease target mRNA levels. Nature. 2010 Aug 12;466(7308):835-40.
2. Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved
targets of microRNAs. Genome Res. 2009 Jan;19(1):92-105.
3. Park SM, Gaur AB, Lengyel E, Peter ME. The miR-200 family determines the
epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1
and ZEB2. Genes Dev. 2008 Apr 1;22(7):894-907.
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