The seed sequence is necessary but insufficient for downregulation of target genes by miR-608

Abstract

MicroRNAs are small, non-coding RNAs that inhibit gene expression posttranscriptionally through interaction with the 3′ untranslated region (3′ UTR) of target mRNAs. The most important factor for downregulation of target genes by miRNA is the “seed region,” which encompasses nucleotides 2–7 at the 5′ end of the miRNA. In this study, sequence determinants for efficient downregulation of target genes were investigated by employing growth-suppressive miR-608 and miR-4651, which shares the seed sequence with miR-608. Cell growth experiments revealed that miR-4651 and miR-608-scram in which the seed sequences were mutated did not inhibit the growth of A549 cells in contrast to wild-type miR-608, which significantly inhibited cell growth. When similarity to miR-608 was increased by replacing sequences of miR-4651 with that of miR-608, cell growth was gradually more inhibited. Similar results were obtained from a luciferase reporter assay using a reporter plasmid containing the 3′ UTR of BCL2L1 and from western blot analysis of BCL2L1, CCND3, and phosphoinositide 3-kinase regulatory subunit 2. Moreover, microarray analyses revealed that overexpression of miR-4651 and miR-608-scram resulted in inefficient downregulation of target genes, and the number of downregulated genes was increased when transfected with MT-3 mimic, which differs from miR-608 by four nucleotides located in the central region. Together, our findings provide a basis for understanding the mechanism underlying target recognition and/or downregulation of target genes by miR-608 and indicate that in addition to seed sequence, central and 3′ parts of miR-608 play an important role in mediating efficient downregulation of target genes.