AROs knock down KRT14 expression in an hnRNPA1 specific manner.
To test whether AROs can knock down gene expression in a targeted
manner, we created a 23 base-pair antisense RNA molecule with homology
to exon 7 of the KRT14 gene and fused an hnRNPA1 recruiting loop to
either the 5’ end, the 3’ end or both sides of the targeting
oligonucleotide. KRT14 was chosen as a knockdown target because it has
been implicated as a driver of the epithelial-mesenchymal transition in
numerous cancers, (Bilandzic et al., 2019, p. 14; Papafotiou et al.,
2016, p. 14) and Exon 7 was chosen due to its small size (47 base-pairs)
and because it contains several motifs for ASF/SF2 exonic splice
enhancer binding, a regulatory element that hnRNPA1 has been shown to
repress (Cartegni et al., 2003; Kashima et al., 2007). As controls, we
also tested an oligonucleotide containing only the 23 bp targeting
domain (i.e. an “antisense only” control with no A1 recruiting loop)
and an ARO with a scrambled targeting domain (Fig 2A), which
respectively served to determine whether the targeting oligonucleotide
or hnRNPA1-recruiting loop alone influenced KRT14 expression. Human
keratinocytes, HaCat cells, were chosen as a model cell line for initial
experiments due to their robust expression of KRT14.
Because hnRNPA1 binds cooperatively (Jean-Philippe et al., 2013), we
hypothesized that we would see the greatest knockdown of gene expression
with the ARO containing two flanking hnRNPA1 recruiting loops
(ARL-Oligo-ARL). Consistent with our expectations, we observed a 56%
knockdown of KRT14 gene expression in ARL-Oligo-ARL compared to the
carrier control (P <= 0.001), and a 53% knockdown of
expression compared with the targeting oligo only (Oligo) control (P
<= 0.01) indicating that the hnRNPA1 recruiting loop was
required for knockdown to occur (Figure 2B). We also saw significant
knockdown of the ARL-Oligo (P <= 0.05) and Oligo-ARL (P
<= 0.05) experimental conditions (18% and 29% respectively),
when compared to the targeting oligo only (Oligo) control.
Interestingly, we observed a small (15%) but statistically significant
difference between the targeting oligo only control (Oligo) and the
scrambled targeting oligo control attached to an hnRNPA1 recruiting loop
(scrOligo-ARL) indicating that the presence of the ARL alone may have a
minor knockdown effect on KRT14. Alternatively, it is possible that the
scrambled targeting oligo has off-target effects influencing the
expression of other transcripts which affect the expression of KRT14.
To test whether the effects of the AROs were hnRNPA1 dependent, each
condition was repeated in the presence of two validated siRNAs designed
to knockdown hnRNPA1 (Figure 2B). Concurrent loss of hnRNPA1 with
expression of ARL-Oligo, Oligo-ARL, and ARL-Oligo-ARL caused significant
and complete loss of previously seen knockdown effects on KRT14
transcript (P <= 0.05, P <= 0.01 and P <=
0.01 respectively). In contrast, differences between the control
constructs, scrOligo-ARL and Oligo, and their hnRNPA1 siRNA knocked down
counterparts were not significant.
Taken together, our results demonstrate that AROs targeting KRT14 knock
down mRNA expression in a targeted and hnRNPA1 dependent manner.