RESUMO
Objective:To investigate the influence of long non-coding RNA-CRNDE in stem cell properties and their mechanism in glioma.Methods:The CD133 +U251 stem cells (U251s) were isolated from human glioma cell line U251 by immunomagnetic beads. Cells were divided into U251s group, U251s-CRNDE group, U251 group, and U251-CRNDE group; CRNDE shRNA lentiviral vectors were transfected into cells in the U251s-CRNDE group and U251-CRNDE group to down-regulate the CRNDE expression. The CRNDE mRNA expression was detected by real-time fluorescent quantitative PCR (RT-qPCR); CCK-8 assay was used to detect the cell viability; Transwell assay was used to detect the cell invasion; wound healing method was used to detect the cell migration; plate cloning assay was employed to detect the clone formation; flow cytometry was used to detect the cell cycles; Western blotting was used to detect the expressions of A2B5, CD133, nestin, Sox2, Oct-4 and Nanog, as well as phosphatidylinositol kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) signal pathway key proteins (PI3K, AKT, phosphorylated-AKT, and mTOR). Results:The CRNDE mRNA expression in U251s group was significantly higher than that in U251 group ( P<0.05). As compared with U251s group, U251s-CRNDE group had significantly decreased cell viability, cell invasion and cell migration, statistically smaller number of cell colony, significantly decreased proportion of cells in G2/M phase, significantly increased proportion of cells in G1/G0 phase ( P<0.05); and the same trend was noted between U251 group and U251-CRNDE group. As compared with U251s group, U251s-CRNDE group had significantly decreased expressions of CD133, nestin, Sox2, Oct-4, PI3K, AKT, phosphorylated-AKT, and mTOR ( P<0.05). Conclusion:The CRNDE expression is increased in glioma stem cells, and down-regulation of CRNDE expression can inhibit the differentiation, metabolism and proliferation of glioma stem cells; and this effect is related to the regulation of PI3K-AKT-mTOR signaling pathway.
RESUMO
Atmospheric (in vitro) oxygen pressure is around 150 mm Hg (20% O₂), whereas physiologic (in vivo) oxygen pressure ranges between 5 and 50 mm Hg (0.7–7% O₂). The normoxic environment in cell culture does not refer to a physiological stem cell niche. The aim of this study is to investigate the effect of oxygen concentration on cell properties of human mesenchymal stem cells (MSCs). We analyzed cell proliferation rate, senescence, immunophenotype, stemness gene expression and differentiation potency with human urine stem cells (USCs), dental pulp stem cells (DPSCs), amniotic fluid stem cells (AFSCs), and bone marrow stromal cells (BMSCs). USCs, DPSCs, AFSCs and BMSCs were cultured under either 5% O₂ hypoxic or 20% O₂ normoxic conditions for 5 days. MSCs cultured under hypoxia showed significantly increased proliferation rate and high percentage of S-phase cells, compared to normoxic condition. In real-time PCR assay, the cells cultured under hypoxia expressed higher level of Oct4, C-Myc, Nanog, Nestin and HIF-1α. In immunophenotype analysis, MSCs cultured under hypoxia maintained higher level of the MSC surface markers, and lower hematopoietic markers. Senescence was inhibited under hypoxia. Hypoxia enhances osteogenic differentiation efficiency compared to normoxia. Hypoxia showed enhanced cell proliferation rate, retention of stem cell properties, inhibition of senescence, and increased differentiation ability compared to normoxia.