shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [miRNA-seq]

miRNA overexpression strategy based on RNA Polymerase (Pol) III mediated expression of short hairpin RNA (shRNA) coding particular miRNA is widely used for miRNA functional studies. For some miRNAs, e.g., encoded in the genome as a part of a polycistronic miRNA cluster, it is most likely the only way for their individual stable overexpression. Here we have revealed that extra \texttt{U} residues (up to five) added Pol III at the 3$'$-end of the transcribed shRNA associated with a shift in the Dicer cleavage position of the shRNA. This results in the undesirable formation of 5$'$-end miRNA isoforms (5$'$-isomiRs), which have a significantly altered seed region compared to the initially encoded canonical hsa-miR-93-5p. The predominant expression of 5$'$-isomiRs without three or four first nucleotides instead of the canonical isoform could be disclosed based on miRNA-Seq analysis. Moreover, mRNA sequencing data showed that the 5$'$-isomiRs of hsa-miR-93-5p presumably regulate their own mRNA targets. Thus, omitting miRNA-Seq analysis may lead to erroneous conclusions regarding revealed mRNA targets and possible molecular mechanisms in which studied miRNA is involved. PC-3 (prostate cancer) cell line was transduced by lentiviral particles coding one of the three types of shRNA to achieve stable overexpression of hsa-miR-93-5p. All shRNA constructs contained the sequence of the canonical hsa-miR-93-5p and differed in their loop sequence only (5'-CUACCUGCACGAACAGCACUUUG-3' -- upper stem; 5'-CUCGAG-3', 5'-UUCAAGAGA-3', 5'-UUCUAAAA-3' -- stem loops; 3'-...UUGAUGGACGUGCUUGUCGUGAAAC-5' -- lower stem). To comprehensively understand the overexpressed isoforms of hsa-miR-93-5p and their impact on the cellular transcriptome profile, we conducted sequencing of both miRNAs (3 repeats for the control, 3 repeats for the first loop and 2 repeats for other ones) and mRNAs (3 repeats for control and all loops).