Some limitations of this study should be noted. First, bulk RNA-seq cannot evaluate the functional relevance of miRNA–mRNA pairing at the cellular level. Since RNA samples for the transcriptome analysis were extracted from homogenized brain tissues, AUD-associated miRNA and mRNA expression changes may not occur in the same type of cells. To identify AUD-associated and cell type-specific miRNA–mRNA pairs, single-cell (or nucleus) RNA-seq can be applied to map miRNA and mRNA transcriptomes at the individual cell level. Second, the functional role of AUD-associated miRNA–mRNA networks in regulating neuronal function was not investigated. We only predicted by bioinformatics programs or based on published studies that a number of AUD-related pathways were regulated by AUD-associated and negatively correlated miRNA–mRNA pairs. Animal model studies can be conducted to determine the influence of miRNA–mRNA interactions on neuronal function and addiction-related behaviors. Third, the transcriptome analysis of postmortem brain tissues cannot determine whether the dysregulation of brain miRNAs and mRNAs was due to pre-existing vulnerability factors (such as genetic variants and/or environmental insults) or long-term alcohol consumption. We intended to verify AUD-associated brain miRNA and
