title: Inhibition of GLI1 Expression by Targeting the CRD-BP-GLI1 mRNA Interaction Using a Specific Oligonucleotide.
abstract: The stabilization of glioma-associated oncogene 1 (GLI1) mRNA by coding region determinant binding protein (CRD-BP) through the Wnt/β-catenin signaling pathway is implicated in the proliferation of colorectal cancer and basal cell carcinoma. Here, we set out to characterize the physical interaction between CRD-BP and GLI1 mRNA so as to find inhibitors for such interaction. Studies using CRD-BP variants with a point mutation in the GXXG motif at each KH domain showed that KH1 and KH2 domain are critical for the binding of GLI1 RNA. The smallest region of GLI1 RNA binding to CRD-BP was mapped to nucleotides (nts) 320-380. A 37-nt S1 RNA sense oligonucleotide, containing two distinct stem-loops present in nts 320-380 of GLI1 RNA, was found to be effective in blocking CRD-BP-GLI1 RNA interaction. Studies using various competitor RNAs with modifications to S1 RNA oligonucleotide further displayed that both the sequences and the structure of the two stem-loops are important for CRD-BP-GLI1 RNA binding. The role of the two-stem-loop motif in influencing CRD-BP-RNA interaction was further investigated in cells. The 2'-O-methyl derivative of the S1 RNA oligonucleotide significantly decreased GLI1, c-myc, and CD44 mRNA levels, in a panel of colon and breast cancer cells. The results from this study demonstrate the potential importance of the two-stem-loop motif as a target region for the inhibition of the CRD-BP-GLI1 RNA interaction and Hedgehog signaling pathway. Such results pave the way for the development of novel inhibitors that act by destabilizing the CRD-BP-GLI1 mRNA interaction.
authors.affiliation: Chemistry Program, University of Northern British Columbia, Prince George, British Columbia, Canada.
authors.forename: Kashif
authors.initials: K
authors.lastname: Mehmood
authors.affiliation: Chemistry Program, University of Northern British Columbia, Prince George, British Columbia, Canada.
authors.forename: Daud
authors.initials: D
authors.lastname: Akhtar
authors.affiliation: Chemistry Program, University of Northern British Columbia, Prince George, British Columbia, Canada.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Mackedenski
authors.affiliation: Chemistry Program, University of Northern British Columbia, Prince George, British Columbia, Canada.
authors.forename: Chuyi
authors.initials: C
authors.lastname: Wang
authors.affiliation: Chemistry Program, University of Northern British Columbia, Prince George, British Columbia, Canada chow.lee@unbc.ca.
authors.forename: Chow H
authors.initials: CH
authors.lastname: Lee
mesh_terms: D020816:Amino Acid Motifs; D001483:Base Sequence; D001665:Binding Sites; D001943:Breast Neoplasms / Q000235:genetics; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D018960:Hyaluronan Receptors; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000494:pharmacology*; D017354:Point Mutation / Q000235:genetics; D000072417:Protein Domains; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000071676:Zinc Finger Protein GLI1 / Q000235:genetics* / Q000378:metabolism
chemical_list: C497483:CD44 protein, human; D018960:Hyaluronan Receptors; C406144:IGF2BP1 protein, human; D009841:Oligonucleotides; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000071676:Zinc Finger Protein GLI1