Abstract:
Glioblastoma multiforme (GBM) is considered the most aggressive brain malignancy associated with poor prognosis. MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression and have been found to play a role in GBM progression. MiRNAs can serve as promising diagnostic and prognostic biomarkers, as well as targets for novel therapies. Additionally, β-amino carbonyl compounds are a class of synthetic molecules with potential anti-cancerous properties that might provide an alternative management strategy for GBM patients. This study sought to investigate the action of a new β-amino carbonyl compound, N-(4-Benzyloxybenzylidene)-2-bromo-4-methylaniline (SHG-8), and its effects upon miRNA regulation within GBM cell models.
In silico analysis was performed to identify the most deregulated miRNAs and their mRNA targets in GBM. Quantitative real-time polymerase chain reaction (RT-qPCR) was carried out to verify the expression profiles of selected miRNAs/mRNA targets. In vitro assays including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), cellular reactive oxygen species (ROS), colony forming, and wound healing were performed to assess the proliferative and migratory properties of U87MG cells prior- and post-exposure to SHG-8. DAPI staining was conducted to examine apoptosis. Small RNA-sequencing (sRNA-seq) was performed to identify the miRNAs that SHG-8 compound activates or supresses. Immunohistochemistry was carried out to evaluate CORO1C protein expression across brain cancer patients’ samples. The absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of SHG-8 were predicted with admetSAR 2.0 using the canonical SMILES with the option ‘ADMET properties for drug discovery’ selected.
In silico analyses suggested that CORO1C was a direct target of miR-21 and miR-128a. RT-qPCR demonstrated that CORO1C was significantly downregulated within GBM cells upon 24h treatment with SHG-8, whilst sRNA-seq revealed significant downregulation of miR-21 and miR-128a. SHG-8 induced significant inhibition of cell viability at 80μM and 100μM. ROS were released following 30min incubation with 50μΜ of SHG-8. Presence of apoptotic bodies were observed post-treatment with 100μΜ SHG-8 in comparison to dimethyl sulfoxide (DMSO). SHG-8 (40μΜ) mediated 94% and 93% open wounding areas at 24h and 48h post-treatment respectively compared to wound openings of DMSO; 1.6% and 0.36%, respectively. In total, 75% of the GBM cases exhibited moderate to high CORO1C expression, similar to 70% of medulloblastoma cases, which sustained strong expression levels. Bioinformatics analysis revealed that SHG-8 demonstrated absorption and distribution features comparable with ibuprofen and was able to cross the blood-brain-barrier.
Synthetic molecules provide a future opportunistic approach for the putative treatment of GBM via miRNA targeting. SHG-8 demonstrated favourable bioavailability and potential to modify the levels of GBM-associated miR-21 and miR-128a. These miRNAs could downregulate CORO1C, subsequently inhibiting tumorigenic processes such as proliferation and migration and alter the malignancy outcome. The high expression levels of CORO1C observed through immunohistochemistry further supported its involvement in highly metastatic brain tumours and thus offers a potential target for future therapies. SGH-8 has shown promising preliminary in vitro results, and hence will be further investigated in regard to its effects upon miRNA regulation.
Audience Take Away Notes:
We synthesized an in-house beta-amino ketone compound (SHG8) that kills glioblastoma cells
SHG-8 demonstrated favourable bioavailability and potential to modify the levels of GBM-associated miR-21 and miR-128a
MiR-21 and miR128 could downregulate CORO1C, subsequently inhibiting proliferation and migration and alter the malignancy outcome
High expression levels of CORO1C observed through immunohistochemistry indicated its involvement in highly metastatic brain tumours and thus offers a potential target for future therapies
SHG-8 demonstrated absorption and distribution features comparable with ibuprofen and was able to cross the blood-brain-barrier
