Knocking down KRT14 via AROs in squamous cell carcinoma reduces cancer migration capability.
Having demonstrated that we can knock down genes via delivery of oligonucleotide AROs, we next sought to test if ARO-mediated gene knockdown could achieve a therapeutic/biologically functional effect. KRT14 expression has been shown to be important for metastasis and invasion in several cancers including bladder and ovarian cancers (Bilandzic et al., 2019, p. 14; Papafotiou et al., 2016, p. 14). SCC9 is a squamous cell carcinoma line used as a model for epithelial-mesenchymal transition studies in head and neck cancer (Zhu et al., 2012). We hypothesized that knocking down KRT14 in SCC9 using our ssRNA anti-KRT14 ARO would reduce the ability of these cells to invade surrounding tissues.
To test this hypothesis, we lipofected SCC9 cells with a KRT14 targeting ARO (ARL-Oligo) or, as a positive control, an siRNA against KRT-14, at 100 nM and assayed RNA expression by RT-qPCR. After 48 hours a near complete knockdown of KRT14 transcript levels was observed in both conditions (P <= .001) (Figure 4B). We also sought to verify knockdown of Cytokeratin-14, the protein encoded by KRT14. We performed a western blot (Figure 5C) and observed a 38% reduction of protein in the siRNA treated cells (P<=0.01) and a 24% knockdown in cells treated with ssRNA anti-KRT14 ARO (P<=0.05). While this knockdown was less than the observed RNA reduction, this is likely due to two factors: the long doubling time of SCC9 (~72 hours) contributing to slow dilution of the protein, and the protein turning over at a slower rate than the RNA, which is actively being degraded in both conditions. Additionally, we observed that differences in protein knockdown between the ARO and siRNA were not significant.
We next sought to assay the invasive potential of SCC9 cells after the loss of KRT14. 48 hours after lipofection with synthesized oligonucleotides or siRNAs, cells were seeded into a Boyden chamber to test their ability to invade into surrounding tissues. This assay uses a transwell that is sealed on one side with a porous membrane coated with Matrigel. The transwell is then suspended inside a larger well, and only invasive cells are able to migrate through the Matrigel. 48 hours after transfection, cells were seeded and incubated for 24 hours. Cells that invaded the Matrigel were then stained, observed via light microscopy (Figure 5B), and counted. We observed a significant decrease in invasive potential of cells treated with both the anti-KRT14 siRNA control (P<=0.05) as well as our ARO (P<=0.05) compared to cells treated only with lipofectamine carrier. Cells treated with the KRT14 siRNA had 39% fewer cells migrate into matrigel than the carrier control, while cells treated with the anti-KRT14 ARO had 43% fewer cells relative to the carrier (Figure 5A).
The above results support two important conclusions. First that the knockdown of KRT14 negatively affects the ability of squamous cell carcinoma to invade surrounding tissues. Second that AROs delivered as ssRNAs are biologically functional and able to knock down protein levels in addition to RNA. We believe these experiments show the usefulness of AROs as novel and useful biological tools for both RNA and protein perturbation.