shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [mRNA-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 mRNAs (3 repeats for control and all loops) and miRNAs (3 repeats for the control, 3 repeats for the first loop and 2 repeats for other ones).

基于RNA聚合酶（RNA Polymerase, Pol）III介导表达编码特定microRNA（miRNA）的短发夹RNA（short hairpin RNA, shRNA）的miRNA过表达策略，已被广泛应用于miRNA功能研究领域。对于部分miRNA而言——例如那些作为多顺反子miRNA簇的一部分由基因组编码的miRNA——这大概率是实现其单基因稳定过表达的唯一可行途径。本研究揭示，RNA聚合酶III在转录所得shRNA的3'端添加的额外尿嘧啶（U）残基（最多可达5个），会引发shRNA的Dicer酶切割位点发生偏移。这会导致非预期的5'端miRNA异构体（5'-isomiRs）形成，与初始编码的经典hsa-miR-93-5p相比，这类异构体的种子区发生了显著改变。通过miRNA测序（miRNA-Seq）分析，可检测到优势表达的5'-isomiRs为缺失前3或4个核苷酸的亚型，而非经典亚型。此外，mRNA测序数据显示，hsa-miR-93-5p的5'-isomiRs大概率会调控其对应的mRNA靶基因。因此，若省略miRNA-Seq分析步骤，可能会对所鉴定的mRNA靶基因及研究所涉miRNA的潜在分子机制得出错误结论。本研究使用编码三种shRNA之一的慢病毒颗粒转导PC-3（前列腺癌）细胞系，以实现hsa-miR-93-5p的稳定过表达。所有shRNA构建体均包含经典hsa-miR-93-5p的序列，仅茎环序列存在差异：5'-CUACCUGCACGAACAGCACUUUG-3'（上游茎区）；5'-CUCGAG-3'、5'-UUCAAGAGA-3'、5'-UUCUAAAA-3'（茎环序列）；3'-...UUGAUGGACGUGCUUGUCGUGAAAC-5'（下游茎区）。为全面解析hsa-miR-93-5p的过表达亚型及其对细胞转录组图谱的影响，本研究对mRNA与miRNA均进行了测序：其中对照组与所有茎环组的mRNA设置3次生物学重复；对照组miRNA设置3次重复、第一组茎环miRNA设置3次重复，其余两组茎环miRNA设置2次重复。