MicroRNA-153/Nrf-2/GPx1 pathway regulates radiosensitivity and stemness of glioma stem cells via reactive oxygen species

Abstract

Glioma stem cells (GSCs) exhibit stem cell properties and high resistance to radiotherapy. The main aim of our study was to determine the roles of ROS in radioresistance and stemness of GSCs. We found that microRNA (miR)-153 was down-regulated and its target gene nuclear factor-erythroid 2-related factor-2 (Nrf-2) was up-regulated in GSCs compared with that of non-GSCs glioma cells. The enhanced Nrf-2 expression increased glutathione peroxidase 1 (GPx1) transcription and decreased ROS level leading to radioresistance of GSCs. MiR-153 overexpression resulted in increased ROS production and radiosensitization of GSCs. Moreover, miR-153 overexpression led to decreased neurosphere formation capacity and stem cell marker expression, and induced differentiation through ROS-mediated activation of p38 MAPK in GSCs. Nrf-2 overexpression rescued the decreased stemness and radioresistance resulting from miR-153 overexpression in GSCs. In addition, miR-153 overexpression reduced tumorigenic capacity of GSCs and increased survival in mice bearing human GSCs. These findings demonstrated that miR-153 overexpression decreased radioresistance and stemness of GSCs through targeting Nrf-2/GPx1/ROS pathway.

MiR-146b-5p overexpression attenuates stemness and radioresistance of glioma stem cells by targeting HuR/lincRNA-p21/β-catenin pathway

“…In order to identify whether miR-146b-5p targets HuR by binding to its 3′ UTR sequence (Figure 4B), we employed luciferase reporter assays. Generally, a declined luminescence indicated that the 3′-UTR was effectively bound and targeted by the miRNA [33, 34]. As shown in Figure 4C, after miR-146b-5p oligos were transfected into GSCs with the wild type reporter construct pGL3-Luc-HuR, luciferase activity was significantly decreased compared with scramble oligos transfection, whereas mutations in predicted target site of 3′-UTR of HuR gene abrogated the inhibition by miR-146b-5p oligos.…”

“…The human glioma cell lines U87 were purchased from the Type Culture Collection of the Chinese Academy of Sciences and cultured in DMEM/F12 medium (1:1, Hyclone) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) in a humidified atmosphere containing 5% CO2 at 37°C. The human GSCs culture U87s, derived from glioma cell line U87, were enriched using serum-free clone formation method with stem cell medium, which has been previously described [33]. The patient-derived GSCs culture SU-2 and non-GSCs glioma cell culture NSSU-2, isolated from a surgical specimen of a 52-year-old female patient with glioblastoma, were kindly provided by Dr. Ting Sun in neurosurgery laboratory of the first affiliated hospital of Soochow University [33, 47, 48].…”

“…The human GSCs culture U87s, derived from glioma cell line U87, were enriched using serum-free clone formation method with stem cell medium, which has been previously described [33]. The patient-derived GSCs culture SU-2 and non-GSCs glioma cell culture NSSU-2, isolated from a surgical specimen of a 52-year-old female patient with glioblastoma, were kindly provided by Dr. Ting Sun in neurosurgery laboratory of the first affiliated hospital of Soochow University [33, 47, 48]. GSCs were cultured as spheres in serum-free DMEM/F12 supplemented with 2% B27 Neuro Mix (invitrogen), 20 ng/mL epidermal growth factor (EGF) and 10 ng/mL basic fibroblast growth factor (bFGF) (PeproTech).…”

“…mRNA expression was analyzed using the SYBR Green PCR Master Mix (Applied Biosystems) on a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA) using standard conditions as previously described [33]. Fold changes in mRNA expression were quantified with the 2 −ΔΔCT relative quantification method using β-actin as house keeping control.…”

“…GSCs with stable integration of the miR-146b-5p mature sequence or scramble sequence were generated through lentiviral-mediated gene transfer [33]. To generate the respective viruses, 293T cells were transfected with the lentiviral vector, pGLV-miR-146b-5p-GFP or pGLV-scr-GFP, and the packaging plasmid PG-P1-VSVG, PG-P2-REV and PG-P3-RRE according to standard protocols.…”