In the past two decades, extensive research effort has been devoted to discover non-coding regulatory RNAs and study their functions. In particular, microRNAs (miRNAs), mainly 20–22 nucleotide (nt) small RNAs (sRNAs), are emerging as an important class of endogenous gene regulators acting at the post-transcriptional level. Mature miRNAs are processed from much longer primary transcripts, called pri-miRNAs, via stem-loop-structured intermediates called pre-miRNAs, and are usually incorporated into the RNA-induced silencing complex where they interact with their target transcripts. It is well established that miRNAs widely exist in animals and plants, and are crucial for many fundamental biological processes.

The replicated tissue samples enabled a systematic examination of the expression patterns of lettuce miRNAs. Normalized by reads per million (RPM), the complete expression profile was obtained. MiRNAs varied in their expression patterns, and some were consistently highly expressed in different tissues, while some varied significantly between different tissues. As shown in the heat map, the expression patterns of 157 miRNAs in four tissues were revealed. miRNA IDs with green, blue, and gray colors represent conserved, Asteraceae-specific, and lettuce-specific miRNAs, respectively. R1 and R2 indicate two samples from root tissue while S, L, and F represent samples from stem, leaf and flower, respectively.

The regulatory elements of miRNAs and target genes do not exist independently, and further constitute a regulatory network. Taking into account the particularity of TFs, that is, they are important miRNA targets that can, in return, regulate miRNA expression, a regulatory network was explored, with TFs, miRNAs, and targets as basic elements. In detail, 1,051 interactions were found between TFs and miRNAs, named TMIs, and 73,133 interactions identified between TFs and miRNA targets, so called TTIs. Together with the 6,540 interactions predicted between miRNAs and targets (MTIs), 46,083 cascades binding TMIs and MTIs were achieved, and 43,554 FFL motifs were constructed.