Figure 3.
AGO CLIP-based design of anticancer mi/siRNAs suppressing cervical cancer with HPV18 infections
(A) AGO-bound regions (BC ≥ 2) of E6/E7 transcript in HeLa cells (GenBank: M20324) represented with target sites of putative antitumor mi/siRNAs, based on target sites of tumor-suppressing miRNAs identified in Figures 1 and 2. (B) Sequences of miR-1, miR-206, and 206/E7 (a mi/siRNA designed based on the miR-1/206 site) derived from the AGO-associated region of the E6/E7 transcript. (C) Luciferase reporter assays with the cognate on-target site for estimating IC50 of 206/E7-mediated repression under different concentrations, compared with transfection of E6 siRNA (E6). Repression of E7 protein in HeLa cells was also examined by immunoblotting with β-actin as a control, relative to NT transfection (upper right inset). −, non-transfected HeLa cells. (D) On-target repression measured by qRT-PCR for E6 and E7 mRNA in HeLa cells; relative expression was normalized by GAPDH mRNA. Notably, suppression of E6 and E7 mRNAs was observed from both E7 and 206/E7. (E) miRNA-like activity of 206/E7 was measured by luciferase reporters with miR-206 seed sites (three 8-mer sites, positions 1–8) compared with miR-206, as performed in (C). (F) Silencing of miR-206 targets by 206/E7 transfection in HeLa cells, examined by qRT-PCR of the miR-206 targets, PTK9, and ADAR1. “206,” miR-206. Of note, comparable suppression was shown in the presence of 206/E7 as much as miR-206. (G) Sequences of miR-218 and 218/E7 where mi/siRNAs were delineated from the AGO-miR-218 site in the E6/E7 transcript (E6/E7). (H and I) qRT-PCR measurement of on-target E7 mRNA (H) and miR-218 target, ROBO1 mRNA (I), in HeLa cells. “218,” miR-218. (J) Sequences of miR-497 and 497/E6, mi/siRNAs derived from the predicted miR-497 site in the E6/E7 transcript. Notably, the corresponding miR-497 site had no overlap with any AGO CLIP reads. (K and L) qRT-PCR experiments for on-target E7 mRNA (K) and miR-497 target, SSRP1 mRNA (L). “497,” miR-497. Intriguingly, 497/E6 transfection only silenced SSRP1 but had no effect on E7 mRNA in HeLa cells. (M) Level of E6/E7 transcript in the presence of miR-218 or miR-497 in HeLa cells, quantitated by qRT-PCR. All p values were from two-sided t tests, relative to NT; ∗p < 0.05; n ≥ 3, repeated with biologically independent samples. Graphs show mean and error bars show SD unless otherwise indicated.