Glioblastoma is a lethal and incurable cancer. Tumor suppressor
miRNAs are promising gene therapy tools for cancer treatment. In silico,
we predicted miR-424 as a tumor suppressor. It had several target genes
from the epidermal growth factor receptor (ERBB) signaling pathway that
are overactive in most glioblastoma cases. We overexpressed miR-424 by
lentiviral transduction of U-251 and U-87 glioblastoma cells confirmed
with fluorescent microscopy and real-time quantitative PCR (qRT-PCR).
Then the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) proliferation assay and scratch wound migration assay were
performed to investigate the miR-424 tumor suppressor effect in
glioblastoma. miR-424s effect on glioblastoma apoptosis and cell-cycle
arrest was verified using Annexin V- phosphatidylethanolamine (PE) and
7-minoactinomycin D (7-AAD) apoptosis assay and cell-cycle assay.
miR-424 predicted target genes mRNA and protein level were measured
after miR-424 overexpression in comparison to the control group by
qRT-PCR and western blotting, respectively. We confirmed miR-424 direct
target genes by dual-luciferase reporter assay. miR-424 overexpression
significantly suppressed cell proliferation and migration rate in
glioblastoma cells based on the MTT and scratch assays. Flow cytometry
results confirmed that miR-424 promotes apoptosis and cell-cycle arrest
in glioblastoma cells. Predicted target genes of miR-424 from the ERBB
pathway were downregulated by miR-424 overexpression. qRT-PCR and
western blotting showed that KRAS, RAF1, MAP2K1, EGFR, PDGFRA, AKT1, and
mTOR mRNA expression levels and KRAS, RAF1, MAP2K1, EGFR, and AKT1
protein level, respectively, had significantly decreased as a result of
miR-424 overexpression in comparison to the control group.
Dual-luciferase reporter assay confirmed that miR-424 directly targets
RAF1 and AKT1 oncogenes. Overall, miR-424 acts as tumor suppressor miRNA
in glioblastoma cells.