In recent years, miRNAs have attracted scientists’ attention because of their capacity to regulate target genes involved in cancer development and progression. Therefore, their implementation as a tool to suppress cancer cell proliferation and invasion either by induction of apoptosis or cell cycle arrest is raised13, 45, 46. The promising outcome of clinical trials in the application of miRNAs in cancer treatment like anti-miR-122, Miravirsen, has shown the great potentials of miRNAs as therapeutic tools12. Herein we showed that the Lentiviral delivery of miRNAs to glioblastoma cells is very efficient. Stable transduction using Lentiviral delivery provides permanent miRNA gene expression via antibiotic selection methods.
U-251 glioblastoma cell line used in this study is a typical model of glioblastoma tumor cells that carries a wide range of genetic mutations. U-251 is mutant for TP53 (encoding P53, a molecular inducer of apoptosis), CDKN2A (encoding P16 and P14, molecular inducers of cell cycle arrest), PTEN (encoding inhibitor molecule of PI3K), NF1 (encoding inhibitor molecule of RAS), and EGFR (encoding epidermal growth factor receptor)47, 48. EGFR, RAS, and PI3K are members of the ERBB signaling pathway, which is amongst the most overactivated genetic mechanisms in glioblastoma patients, leading to cell proliferation and invasion49. So a miRNA that targets and suppresses the ERBB signaling pathway was predicted to be a potential tumor suppressor miRNA.
Our bioinformatical analysis predicted that miR-429 could target several members of the ERBB pathway. These molecular targets were upregulated in glioblastoma tissues comparing to the normal brain in silico and were also subject to alterations (mutations or copy number variations) in glioblastoma patients. Besides, miR-429 expression was reduced in glioblastoma patients samples. These findings suggested that overexpression of miR-429 in glioblastoma cell lines might modulate some of these oncogenes from the ERBB pathway directly or indirectly, so suppress glioblastoma tumor cells proliferation and migration.
Real-time PCR and western blotting results supported the bioinformatical prediction. PIK3CA, PIK3CB, KRAS, EGFR, MYC, BCL2, PRKCA, and SHC1 mRNAs significantly decreased in Real-time PCR after miR-429 transduction in U-251 cells. Also, significant downregulation of PIK3CA, PIK3CB, KRAS, EGFR, and MYC proteins in western blotting occurred. Other predicted targets were not measured, as these seemed functionally more relevant. Luciferase assay confirmed that EGFR, MYC, and BCL2 are direct targets of miR-429. Other potential targets are not in the luciferase assay. Direct targeting of MYC50–52 and BCL250, 53, 54 by miR-429 was in previous reports for other cancers. Our data confirm this in glioblastoma. MYC is a transcription factor for the ERBB pathway that facilitates the expression of many genes functioning in cell cycle promotion and growth and, BCL2 is an anti-apoptotic agent that promotes survival. Besides, We identified EGFR as a novel target of miR-429.
The EGFR expression becomes higher in 60% of glioblastoma patients. It is a receptor tyrosine kinase and triggers its downstream signal pathways like PI3K/AKT/mTOR and RAS/RAF/MEK55. Once EGFR becomes active by its ligand or constitutively activating mutations like EGFRviii56, it activates other kinases like PI3K, which affects many genes regulation through the activation of AKT oncogene. Activation of the PI3K/AKT/mTOR pathway can lead to glioblastoma tumors expansion and invasion to other tissues49 and may also be a result of mutations in PI3KCA, KRAS, PTEN, or NF127. KRAS activates PI3KCA while PTEN inhibits PI3KCA to phosphorylate AKT, NF1 inhibits KRAS. KRAS is active in most glioblastomas and necessary for the persistence of glioblastoma tumors in mouse models57. It also triggers the oncogenic pathway of MAPK (RAS/RAF/MEK/ERK) in glioblastoma58. So, the downregulation of PIK3CA, PIK3CB, KRAS, EGFR, and MYC proteins by miR-429 in our study, suggests a tumor suppressor effect of miR-429 in GBM cell lines.
Our results for MTT proliferation assay and scratch wound assay of U-251 GBM cells confirmed the tumor suppressor effect of miR-429 in glioblastoma. Although miR-429 suppressed proliferation and migration in U-251 cells, it did not induce apoptosis. So we speculate it may induce differentiation in GBM cells with suppressed proliferation, which is along with our results on the cell-cycle arrest of GBM cells by miR-429 come from flow cytometry analysis of cell-cycle. Our previously published data confirm that the neuronal differentiation genetic markers increase by miR-429 overexpression in GBM cells59. Consistent with our results, Chen et al. showed downregulation of miR-429 happens in glioblastoma patients samples. Thay investigated the suppressor effect of miR-429 overexpression on migration and invasion of U87 GBM cells and confirmed BMK1 as miR-429 direct target60. Dong et al. also showed downregulation of miR-429 in glioblastoma, and they could induce apoptosis and suppress proliferation and invasion in U251 and U87 GBM cells by miR-429 mimics. They confirmed SOX2 as a miR-429 direct target61. Although their finding on apoptosis induction by miR-429 is inconsistent with our result, the whole conclusion on the tumor suppressor role of miR-429 in glioblastoma cells is similar. The disagreeing results may come from different delivery methods of miR-429. However, We did not find any significant increase in the apoptosis rate after miR-429 lentiviral transduction to U-251 cells. Otherwise, we present the miR-429 arresting cell-cycle as a new perspective.
Besides, our approach to using miRNAs to modulate a genetic pathway instead of a unique target gene, especially in genetically heterogeneous cancers like glioblastoma, seems to make the bioinformatical prediction more realistic and efficient in vitro or in vivo. Our findings, especially the miR-429 regulatory effect on the ERBB signaling pathway in glioblastoma, suggests its potential role as a therapeutic agent not only in glioblastoma but also in any other cancer or diseases, in which ERBB signaling pathway over activation is the main factor.