Inhibition of GLI1 Expression by Targeting the CRD-BP-GLI1 mRNA Interaction Using a Specific Oligonucleotide

Area of Science:

• Molecular Biology
• Cancer Research
• RNA Biology

Background:

• Coding region determinant binding protein (CRD-BP) stabilizes glioma-associated oncogene 1 (GLI1) mRNA.
• This interaction is crucial for the Wnt/β-catenin signaling pathway and implicated in colorectal cancer and basal cell carcinoma proliferation.
• Targeting this interaction could offer novel therapeutic strategies for cancer.

Purpose of the Study:

• To characterize the physical interaction between CRD-BP and GLI1 mRNA.
• To identify specific regions involved in CRD-BP binding to GLI1 mRNA.
• To develop inhibitors targeting the CRD-BP-GLI1 mRNA interaction.

Main Methods:

• CRD-BP variants with point mutations in KH domains were used to identify critical binding regions.
• RNA-binding mapping identified the smallest CRD-BP binding region on GLI1 mRNA (nucleotides 320-380).
• A 37-nt S1 RNA oligonucleotide, mimicking the identified region, was synthesized and tested for inhibitory effects in vitro and in cancer cells.

Main Results:

• KH1 and KH2 domains of CRD-BP are critical for GLI1 RNA binding.
• A two-stem-loop motif within nucleotides 320-380 of GLI1 RNA is the primary binding site for CRD-BP.
• The S1 RNA oligonucleotide effectively blocked CRD-BP-GLI1 RNA interaction and significantly reduced GLI1, c-myc, and CD44 mRNA levels in cancer cells.

Conclusions:

• The two-stem-loop motif in GLI1 mRNA is a critical target for inhibiting CRD-BP binding.
• The S1 RNA oligonucleotide demonstrates potential as a therapeutic agent by destabilizing the CRD-BP-GLI1 mRNA interaction.
• This study provides a foundation for developing novel inhibitors targeting the Hedgehog signaling pathway via CRD-BP-GLI1 mRNA modulation.