Preliminary identification of the MiR-548ah-5p/THBD axis for heart failure via DirectTarget causal framework and...

Identifying putative direct regulatory interactions between microRNAs (miRNAs) and messenger RNAs (mRNAs) is crucial for understanding the molecular mechanisms of heart failure (HF). This exploratory study aimed to investigate these putative direct interactions within the miRNA-mRNA axis in HF.

We used the GSE135055 dataset to screen for differentially expressed miRNAs (DE-MiRNAs) and mRNAs (DE-mRNAs). To infer putative direct interactions between them, we applied the DirectTarget framework, utilizing the Interventional Calculus when the DAG is Absent (IDA) method for causal inference, combined with target information predicted from sequence data. We then constructed putative direct regulatory networks for the screened miRNA-mRNAs. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the genes in the putative regulatory network. Finally, the miRNA-mRNAs exhibiting the strongest negative regulatory relationships were experimentally validated in vitro using dual luciferase reporter assays, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and western blotting. In addition, we further investigated the hub miRNA-mRNA expression and exploratory discriminatory ability.

Differential expression analysis between HF and control groups identified 31 miRNAs and 163 mRNAs. The resulting regulatory network comprised 348 putative direct miRNA-mRNA axes, involving 27 miRNAs and 116 mRNAs. Notably, the miR-548ah-5p/THBD pair exhibited the strongest negative regulatory relationship, while miR-4422 and TIFA were involved in the most regulatory interactions. GO enrichment analysis revealed that the extracellular matrix (ECM) was the most significantly enriched term. KEGG pathway analysis identified key pathways such as AGE-RAGE, PI3K-Akt, and ECM-receptor interactions. The gene THBD, with its high connectivity in the network was considered a potential key candidate. Our in vitro experiments further confirmed that miR-548ah-5p negatively regulates THBD expression. Receiver operating characteristic (ROC) analysis showed that miR-548ah-5p and THBD exhibit favorable exploratory classification ability for HF.

This study demonstrates the effectiveness of the DirectTarget framework in identifying putative miRNA target interactions, providing a valuable addition to existing approaches. To our knowledge, this is the first report that the miR-548ah-5p/THBD axis is a key regulatory axis in HF, suggesting a potential new avenue for further research.