Model-guided quantitative analysis of microRNA-mediated regulation on competing endogenous RNAs using a synthetic gene circuit

Abstract

Competing endogenous RNAs (ceRNAs) cross-regulate each other at the posttranscriptional level by titrating shared microRNAs (miRNAs). Here, we established a computational model to quantitatively describe a minimum ceRNA network and experimentally validated our model predictions in cultured human cells by using synthetic gene circuits. We demonstrated that the range and strength of ceRNA regulation are largely determined by the relative abundance and the binding strength of miRNA and ceRNAs. We found that a nonreciprocal competing effect between partially and perfectly complementary targets is mainly due to different miRNA loss rates in these two types of regulations. Furthermore, we showed that miRNA-like off targets with high expression levels and strong binding sites significantly diminish the RNA interference efficiency, but the effect caused by high expression levels could be compensated by introducing more small interference RNAs (siRNAs). Thus, our results provided a quantitative understanding of ceRNA cross-regulation via shared miRNA and implied an siRNA design strategy to reduce the siRNA off-target effect in mammalian cells. RNAs that are loaded onto RNA-induced silencing complexes (RISC) and subsequently bind to their target RNAs. In mammalian cells, the perfect pairing of miRNA to target RNAs causes RNA cleavage through the RNA interference (RNAi) pathway, whereas partial pairing results in translational repression and RNA destabilization (1, 2). miRNA-mediated regulation can be triggered by only 6-nt complementarity of the miRNA 5′-end "seed region" to the target RNA, which confers each miRNA species the capacity to interact with multiple RNA species, including gene-coding mRNAs (3, 4), long noncoding RNAs (5), and circular RNAs (6). Similarly, each RNA species can interact with multiple miRNA species through various miRNA response elements (MREs) (7).The complex interaction network of miRNAs and their target RNAs has been shown to allow indirect cross-regulation between different competing endogenous RNAs (ceRNAs) by sequestering shared miRNAs, which is essential for regulating many biological functions (7). The strength of ceRNA regulation is largely determined by the relative abundance and binding strength of ceRNAs and miRNAs and whether the miRNA-bound ceRNA decays through a stoichiometric mechanism or a catalytic mechanism (8-10). The threshold-like behavior of the ceRNA regulation has been experimentally observed by measuring the abundance of two ceRNAs, phosphatase and tensin homolog (PTEN) and vesicle-associated membrane protein (VAMP)-associated protein A (VAPA) across various cell lines (8). Nevertheless, many quantitative predictions deduced from miRNA-ceRNA computational models have not been experimentally validated. Another intriguing question is whether the miRNA-mediated catalytic mechanism can be affected by the miRNA-mediated stoichiometric mechanism through a ceRNA effect or vice versa.Currently, the ability to systematically elucidate features of the ceRNA effect is impeded by th...

Impact of MicroRNA Levels, Target-Site Complementarity, and Cooperativity on Competing Endogenous RNA-Regulated Gene Expression

“…Thus in ESCs, as in hepatocytes (Denzler et al., 2014), ceRNA-regulated gene expression through upregulation or downregulation of a single transcript is unlikely. Similar results have been reported in HEK293 cells (Yuan et al., 2015). …”

ceRNA in cancer: possible functions and clinical implications

“…Moreover, in vivo experiments showed that up to 23-fold increase of ALDOA abundance did not result in detectable miR-122 inhibition or de-repression of miR-122 targets 15. This is in agreement with the result of another study,13 revealing that active ceRNA interplay was observed only when a large number of ectopic ceRNA transcripts were introduced. This is possibly due to that ceRNA need to compete with the whole target pool of the miRNA species to titrate the latter.…”

“…Despite the increasing number of studies that report the phenotypic effects of ceRNA deregulation in cancer, the current ceRNA theory remains controversial regarding to whether they are really active in the endogenous cellular context 13 15…”

The Emerging Function and Mechanism of ceRNAs in Cancer

“…In addition to their utility in predicting ceRNA regulation, the computational methods, especially biophysical and mathematical modeling approaches have provided significant insight into the mechanism of ceRNA crosstalk [34, 63, 64]. Levine et al utilized mass-action equations to quantitatively model ceRNA-like sRNA-mediated RNA–RNA crosstalk in E. coli [34].…”