A Systematic Study On The Role Of CeRNA As An Intrinsic Component Of The EMT Regulatory Circuit

The epithelial-to-mesenchymal transition(EMT)is a fundamental developmental program that has been implicated in metastasis,a detrimental progress contributing to more than 90% of cancer-related deaths.EMT is associated with dramatic changes in the expression of thousands of genes and is characterized by the downregulation of epithelial markers,such as CDH1,and the upregulation of mesenchymal markers,such as CDH2,VIM and FN1.MicroRNAs(miRNAs) are ubiquitous post-transcriptional regulators that impact RNA stability and the rate of translation by pairing to complementary sites(referred to as miRNA response elements,MREs)within target RNAs.The interation between miRNAs and their RNA targets is characterized by a many-to-many relationship in which a single miRNA can repress multiple RNA targets and a single RNA can contain MREs corresponding to multiple miRNAs.Consequently,many RNAs share significant overlap in their MREs.Hence,a competitive endogenous RNA(ceRNA)hypothesis has been proposed;this hypothesis suggests that RNAs can regulate each other by competing for a limited pool of miRNAs.Theoretical analyses have suggested that ceRNA crosstalk may regulate essential biological processes,and studies have reported that functional ceRNAs regulate key biological functions and contribute to diseases such as cancer.However,these studies often lack the absolute quantification of miRNAs and the corresponding ceRNAs,the stoichiometry between which is crucial for ceRNA-based regulation.Consequently,whether the effects of ceRNA exist under physiological conditions has been challenged.Here,we examined the dynamics of ceRNAs during TGF-?-induced EMT.We observed that TGFBI,a transcript highly induced during EMT in A549 cells,acted as the ceRNA for miR-21 to modulated EMT.We further identified FN1 as the ceRNA for miR-200 c in the canonical SNAIL-ZEN1-miR200 circuit in MCF10 A cells.Absolute quantification confirmed that induced TGFBI and FN1 outnumber their target miRNAs.Experimental assay demonstrated that the dynamically induced ceRNAs are critical to the induced of EMT.Moreover,we showed that the stoichiometry between miRNA and ceRNA is critical parameter to determine the reversibility of EMT.These results help to establish the physiological relevance of ceRNA and suggest that ceRNA is an intrinsic component of the EMT regulatory circuit and may repress a universal target to disrupt EMT.