Abstract

 
 
 
 Purpose: To examine the role of miR-557 in the pathogenesis and development of glioblastoma (GBM) and its potential regulatory mechanism.
 Methods: Dysregulation of miR-557 in GBM cells was determined using quantitative (real-time) PCR (qPCR). Cell proliferative ability, migratory activity and invasive ability were measured using the MTT colorimetric assay, wound healing assay and Transwell chambers assay. The direct target mRNA of miR-557 was predicted, and further validated using TargetScan and luciferase assay, respectively.
 Results: Compared with human astrocytes (HAs), the downregulation of miR-557 was observed in human GBM cell lines. In U-251MG and A172 cells that overexpressed miR-557 mimics, the level of proliferation was significantly reduced, the wound width was larger, and the number of invaded cells wan decreased than that of NC mimics. Predictive results from TargetScan and results of luciferase assay demonstrated that miR-557 directly targets the 3’-untranslated region (3’-UTR) of ADAM17. In A172 cells and U-251MG cells, the upregulation E-cadherin (E-cad) and the downregulation of N- cadherin (N-cad) and vimentin (Vim) were caused by miR-557 mimics as compared with NC mimics. Expression of ADAM17, NICD, HES1, and EGFR was downregulated by miR-557 mimics and upregulated by miR-557 inhibitors in A172 cells and U-251MG cells.
 Conclusion: Downregulation of miR-557 accelerates GBM cell growth and malignancy by directly targeting ADAM17 3’-UTR, providing a prognostic and potential therapeutic factor for new drug discovery in GBM.
 
 
 
Highlights
Glioblastoma (GBM) is the most aggressive tumor in the brain and central nervous system [1]
The primary antibodies were: anti-Ecadherin (E-cad) (#3195, 1:500 dilution), anti-Ncadherin (N-cad) (#13116, 1:500 dilution), antivimentin (Vim) (#5741, 1:1000 dilution), antiADAM17 (#ab2051, 1:300 dilution), anti-NICD (#4147, 1:1000 dilution), anti-HES1 (#11988, 1:200 dilution), anti-epidermal growth factor receptor (EGFR) (#3197, 1:800 dilution), and anti-β-actin (#4970, 1:5000 dilution)
Expression of A Disintegrin and metalloproteases 17 (ADAM17), NICD, HES1, and EGFR was decreased by miR-557 mimics, whereas expression of these proteins was increased by inhibition of miR-557 in both
Summary
Glioblastoma (GBM) is the most aggressive tumor in the brain and central nervous system [1]. Downregulation of miR-148 prevented cell proliferation and migration through targeting ITGA9, indicating an inhibitory effect on the development of GBM [13]. The extraction of total RNA from GBM cells was performed, followed by qPCR under conditions of 40 cycles of 95 °C (10 s), 60 °C (30 s), and 72 °C (30 s). The DNA fragments containing the putative miR557 target site within the ADAM17 3’-UTR (wildtype (WT) or mutant (MUT)) was cloned into psiCHECK-2 vector (Promega, Madison, WI, USA). The primary antibodies (anti-ADAM17 was obtained from Abcam, Cambridge, UK, the others were obtained from Cell Signaling Technology, Danvers, MA, USA) were: anti-Ecadherin (E-cad) (#3195, 1:500 dilution), anti-Ncadherin (N-cad) (#13116, 1:500 dilution), antivimentin (Vim) (#5741, 1:1000 dilution), antiADAM17 (#ab2051, 1:300 dilution), anti-NICD (#4147, 1:1000 dilution), anti-HES1 (#11988, 1:200 dilution), anti-EGFR (#3197, 1:800 dilution), and anti-β-actin (#4970, 1:5000 dilution). NC mimics in both U-251MG and A172 cells (Figure 2B)
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