Downregulation of miR-21 and CORO1C in glioblastoma cells treated with a β-amino carbonyl compound
Glioblastoma multiforme (GBM) is an aggressive brain malignancy characterised by its invasive nature. Current treatment has limited effectiveness, resulting in poor patients’ prognoses. β-amino carbonyl compounds (β-ACs) have gained attention due to their potential anti-cancerous properties. In vitro assays were performed to evaluate the effects of an in-house synthesised β-AC compound, named SHG-8, upon GBM cells. Small RNA-sequencing (sRNA-seq) and biocomputational analyses investigated the effects of SHG-8 upon the miRnome and its bioavailability within the human body. SHG-8 exhibited significant cytotoxicity in U87MG and U251MG GBM cells with IC50 values of 85μΜ and 100μΜ, respectively. Significant inhibition of cell migration and proliferation was induced in the presence of SHG-8. GBM cells treated with the compound were seen to release significant amounts of reactive oxygen species (ROS) at 50μΜ and 100μΜ SHG-8 concentrations. Annexin V staining also demonstrated that the compound leads to apoptosis. sRNA-seq revealed a dramatic shift in miRNA expression profiles upon SHG-8 treatment and significant downregulation of miR-7974 and miR-21. Furthermore, miR-3648 was significantly upregulated in the presence of SHG-8. The sequencing analysis also identified that SHG-8 had a negative effect on the regulation of the Wnt/β-catenin pathway. Real-time polymerase chain reaction (RT-qPCR) demonstrated a significant downregulation of CORO1C, a target gene of miR-21. In silico analysis indicated SHG-8's favourable absorption, distribution, and potential to cross the blood-brain barrier. We concluded that SHG-8's inhibitory effects on GBM cells may involve miR-21-mediated CORO1C inhibition.
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Preparation of samples for sRNA-seq U87MG RNA samples were extracted with Trizol. A nanodrop bioanalyzer was used to detect the purity of RNA samples, concentration, and integrity, and to ensure the use of qualified samples for sequencing. RNA integrity was assessed using the RNA Screen Tape Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Samples with RIN numbers greater than seven were sent off for external sRNA-seq to Biomarker Technologies (Biomarker Technologies, Inc., CA, USA). Library preparation for sRNA sequencing A total of 1.5μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext®Ultra™ small RNA Sample Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. Firstly, ligated the 3′ SR Adaptor was made via mixing 3′ SR Adaptor for Illumina, RNA and Nuclease-Free Water. The mixture system was incubated for 2min at 70°C in a preheated thermal cycler. The tube was transferred on ice. Then, 3′ Ligation Reaction Buffer (2X) and 3′ Ligation Enzyme Mix ligate the 3′ SR Adaptor were added, and the mixture was incubated for 1 hour at 25°C in a thermal cycler. To prevent adaptor-dimer formation, the SR RT Primer hybridizes to the excess of 3′ SR Adaptor (that remains free after the 3′ ligation reaction) and transforms the single-stranded DNA adaptor into a double-stranded DNA molecule. Secondly, ligation of the 5′ SR Adaptor was performed, followed by reverse transcription synthetic chain reaction. Lastly, PCR amplification and Size Selection followed. PAGE gel was used for electrophoresis fragment screening purposes and rubber cutting recycling as the pieces get small RNA libraries. PCR products were purified (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v4-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500 platform and paired-end reads were generated. Data analysis Raw data (raw reads) of fastq format were first processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, ploy-N and low-quality reads from raw data, and reads were trimmed and cleaned by removing the sequences smaller than 18 nucleotides or longer than 30 nucleotides. At the same time, Q20, Q30, GC-content and sequence duplication level of the clean data were calculated. All the downstream analyses were based on clean data with high quality.
American University of Ras Al Khaimah