Short GC-rich RNA similar to miR 1909 and 1915 folds in silico with the 5'-UTR and ORF of Notch and responders: Potential for the elimination of cancer stem cells

Abstract

Novel therapeutic approaches to eliminate cancer stem cells (CSCs) are being developed. This development is imperative as CSCs are resistant to drugs; they divide activated by ligands on the epithelium or on neighboring cancer cells. Specific commands for division originate from Notch-1 ligands. Notch-1 cleavage inhibitors can have opposite effects from the ones expected when the levels of Notch ligands are high on neighboring cancer cells. High levels of Jagged-1 are a common feature of ovarian tumors. Some gene pathways enhance, others repress transcription of Notch-1, while Notch-1 itself activates Myc and HIF-1α. RNA-based therapies need effector RNAs (eRNAs) with broad and focused specificity. eRNAs are short RNAs (20-30 nt long) which mediate biological effects. Two to three inhibitory RNAs with high net folding/hybridization/binding (and thereafter folding), and free energy (Net-ΔG) with multiple mRNAs can replace many miRs as eRNAs and overcome the complexity of identification of specific targets for each miR and competitive inhibition on delivery of small amounts of many miRs at the same time. To discover candidate eRNAs with multiple high affinity target sites or sequences (and thereafter targets), we searched for sequences containing more than randomly probable G and C. G and C bind with more hydrogen bonds than the pair A:T. We identified the sequence, Notch-1,33-56 in the ORF of Notch-1 mRNA. Notch-1,33-56 has a GC frame of 2 asymmetrical halves in 24 nucleotides. Each GC group has a different third nucleotide. Since GC is repeated, the third nucleotide defines the specificity as a 'bar code'. The complementary strand to Notch-1,33-56, binds in silico nt at 5'-UTR, ORF and 3'-UTR of mRNA. For simplification, the sequence of Notch-1,33-56 was designated HHN1 and its complementary strand, anti-HHB. We introduced novel quantitative parameters: Net-ΔG and mean Net-ΔG/bond. We quantified the Net-ΔG of folding, in silico, of anti-HHB with additional targets in Notch-1,1-404. The targets of anti-HHB contained 11-12 complementary nucleotides and formed small loops with anti-HHB upon folding. Anti-HHB folded with 3-4 distinct targets in each mRNA from 50 mRNAs. Targets were in 5'-UTR (40%), ORF (50%) and 3'-UTR (10%). Anti-HHB also folded with high Net-ΔG with Notch-1 targets, c-Myc and HIF-1α, suggesting it can inhibit EMT. Human embryonal stem cell (hESC) miRs, 1909 and 1915, folded with Notch-1,8-29 and Notch-1,33-56, respectively with a similar Net-ΔG as anti-HHB. This finding suggested a natural feedback mechanism aiming to inhibit Notch-1 translation which is activated in stem cells by miRs with a similar sequence as anti-HHB, and anti-HHB can be used when the miRs 1915 and 1909 are absent. The consensus sequence of 18 targets folded with HHB with the highest Net-ΔG (range -10.20 to 24.00 Kcal/mol) similar to that of two Drosophila transposons. Targeting 'domesticated transposons' carried by humans with eRNAs may become a universal approach to treat cancer. Anti-HHB is the first candidate eRNA to fold, in silico, with multiple targets in 5'-UTR and ORF of Notch-1 partners with at least 2-times higher ΔG than natural miRs with 3'-UTR Notch-1.