Abstract
MicroRNAs have been found to play a profound role in embryonic and post-natal development through their regulation of processes such as cell proliferation, differentiation, and morphogenesis. The microRNA-30 (miR-30) family is necessary for vertebrate hepatobiliary development; however, the mechanism through which miR-30 regulates these processes is not fully understood. Here, we identify genes directly regulated by miR-30 that have been characterized as key developmental factors. The targets were confirmed via a luciferase reporter assay, following exogenous over-expression of miR-30a and miR-30c2 in cultured cells. Five novel miR-30ac2 targets were identified using this approach, all of which play crucial roles in hepatobiliary development or are involved in hepatocellular carcinoma and cholangiocarcinoma.
Highlights
MicroRNAs are short non-coding RNAs that regulate cell function, differentiation, organ development, and disease states
Using gene expression profiling in cultured hepatoblasts[9], we previously identified a set of mRNAs whose levels are increased following antisense oligonucleotide (ASO)-mediated inhibition of miR30a9
We transfected a human embryonic kidney cell line transformed with the SV40 large T antigen (HEK293FT) with a doxycycline-inducible green fluorescent protein (GFP) and miR-30ac[2] expression plasmid
Summary
MicroRNAs are short non-coding RNAs that regulate cell function, differentiation, organ development, and disease states (reviewed in[1,2,3,4]). MiRNAs regulate gene expression via the RNA-induced silencing complex (RISC), leading to translational repression and/or transcript degradation[5]. The identification of gene targets for individual miRNAs is key to understanding their function, necessitating accurate methods to identify significant mRNA targets. The prevailing methods of miRNA target identification begin with computational algorithms based on free energy change calculations of complementary binding, other sequence features, and evolutionary conservation[6,7]. The data gathered from these computational methods are typically validated by luciferase reporter assays in cultured cells, in which the activity of the relevant miRNA is altered (i.e., by miRNA over-expression or repression)
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