MicroRNAs are small RNA molecules, that are associated with Argonaute proteins enabling them to base pair with complementary target mRNAs. Based on their complementarity, translational repression or silencing of target mRNAs is mediated. MicroRNA-21 (miR-21) is one of the most predominant microRNAs in the mammalian heart and strongly upregulated in failing myocardium. Synthetic inhibitors of miR-21 have been shown to attenuate cardiac dysfunction. Despite the well-known therapeutic effect of antimir-21, the precise mechanism, i.e. the regulated targetome, remains largely unknown. We have recently developed a method to identify high affinity and highly regulated microRNA targets using Argonaute2-ribonucleoprotein immunoprecipitation (AGO2-RIP) in the presence of an antimiR directed against the microRNA of interest followed by next generation RNA sequencing (Werfel et al. Nucl Acids Res 2017). Here, we use this approach to elucidate the miR-21-mRNA network in failing myocardium.

8 weeks-old mice were subjected to a model of pressure overload-induced cardiac hypertrophy and, two weeks after surgery, antimiR-21 was injected intravenously for three days. Two days later, an AGO2-RIP was performed either from the total heart or from the main cardiac cell types (myocytes, fibroblasts, macrophages or endothelial cells). Next generation sequencing of the AGO2-immunoprecipitated mRNA and total RNA followed by bioinformatic analysis identified the most de-enriched (RIP fold change>1, p-value<0.05) and most de-repressed targets (mRNA fold change>1, p-value<0.05) in the antimiR-21-treated group compared to the control group in the total heart and for the main distinct cardiac cell populations. In addition, single cell sequencing allowed us to see subtype-specific antimiR-21 effects of the main cardiac cell types. Genetic deconvolution of bulk RNA sequencing integrating in cell type-specific gene expression analysis allowed us to identify the antimiR-21 effect in different cell types over time.

Taken together, we developed a biochemical/bioinformatic approach based on genetic deconvolution of RNA-Seq of antimiR-treated tissue that allows to define the regulated targetome of a microRNA upon therapeutic manipulation. Our data on miR-21 identified the cardiac cell types, where antimiR-21 exerts its therapeutic effect and the molecular mechanism underlying its effects. Our deconvolution approach should be applicable to the analysis of any microRNA therapeutic intervention as well as to the analysis of general RNA datasets of the heart.