Mir-20/106 Promote Invasion Of Human Glioma Stem Cells By Down-regulating TIMP-2 Expression

Background and Objective:Glioblastoma (GBM) is the most common and lethal malignant primary brain tumor of the central nervous system (CNS). GBM is characterized by invasive growth results in high recurrence after therapies and poor prognosis. Although various cells and molecules have been documented involved in the invasion processes of GBM, the initiating factors remained to be elucidated.More recent data have shown that cancer stem cells (CSCs) may be the primary culprit in tumorigenesis and development of cancer. We have reported that a putative population of glioma stem cells (GSCs) has a capacity of self-renewal, multipotency of differentiation and initiation of tumors. Although the highly invasive nature of glioma cells (GCs) is also implicated in the failure of current therapies, it is not clear whether GSCs are involved in invasiveness of GBM. However, the relevant mechanism remains unknown.miRNAs are approximately 21-23 nucleotides small noncoding RNAs that down-regulate expression of target genes by specifically interacting with 3'untranslated regions (3'UTR), which play a key role in regulating expression of certain oncogenes and tumor suppressor genes. Abnormal expression of miRNA has been linked with many cancers including glioma, and the significance of functional miRNA alteration indicates that miRNAs play roles in cell proliferation, apoptosis, invasion, angiogenesis of glioma. miR-20a and miR-106a separately belong to the miR-17-92 cluster and its paralogous cluster miR-106a-363. miR-20/106 is widely expressed in various types of cells. Studies have suggested that aberrant expression of miR-20/106 works as oncogenes or tumor suppressors. Nonetheless, the role of miR-20/106 in GSC invasion remains unclear.Tissue inhibitor of metalloproteinases-2 (TIMP-2), matrix metalloproteinase 2 (MMP2) and MMP9 play an important role in tumor invasion. Almost all types of ECM can be degraded by MMP2 and MMP9 because of their specific enzymatic activity for certain ECM components. The activation of MMP2 and MMP9 is associated with tumor invasion and poor prognosis. Previous studies have demonstrated that the inhibition of MMP2 and MMP9 could suppress cell invasion. TIMP-2 is a natural inhibitor of MMP2 and MMP9. TIMP-2 is frequently reduced in many tumors including glioma with unknown mechanism. By computational prediction and bioinformatics analysis, TIMP-2 might be one of the target genes regulated by miR-20/106. Therefore, we hypothezed that miR-20/106 might promote invasion of human GSCs by down-regulating TIMP-2 expression. We firstly isolated and characterized GSCs from human GBM cell line U87 and primary human glioma samples. We then examined the expression of miR-20/106 and TIMP-2 pathway in human GSCs and GCs. We further tested the effect of miR-20/106 on human GSCs invasion by miR-20/106 inhibitors.We finally investigated whether miR-20/106 directly regulated TIMP-2 expression.Materials and Methods:1. Fluorescence-activated cell sorting (FACS) technology was utilized to isolate CD133~+ cells from U87 and primary glioma cell population.2. The spontaneous neurospheres formation, immunofluorescence of neural stem cell markers, capability of multilineage differentiation, and in vivo tumorigenicity were tested for characterization for GSCs.3. The microRNA array and real-time quantitative RT–PCR (qRT–PCR) were used to detect the expression of miR-20/106 in human GSCs and GCs.4. qRT–PCR and western blot were used to examine the expression difference of TIMP-2 and its downstream targets MMP2 and MMP9 in human GSCs and GCs.5. Transwell invasion assay was used to detect the invasiveness of GSCs and GCs.6. Transwell invasion assay was used to identify a role for miR-20/106 in promoting invasion of GSCs.7. qRT–PCR and western blot were used to examine the expression of TIMP-2 and its downstream targets MMP2 and MMP9 in human GSCs transfected with miR-20/106 inhibitors.8. Luciferase reporter assay was used to demonstrate miR-20/106 directly regulated TIMP-2 expression.Results:1. Isolation and characterization of GSCs from U87 and primary glioma cells. CD133~+ cell fraction accounted for 0.5%, 2.4% and 1.5% of the total population in U87, and two primary human glioma samples respectively. After FACS sorting, CD133~+ and CD133- population were re-examined for sorting efficiency. We used the CD133~+ cell (purity of above 95%) and CD133- cell (CD133~+ cell below 1%) for our study.The CD133~+ cells were demonstrated to have the capacity of forming spontaneous neurospheres. The tumor cells in the neurospheres expressed both CD133 and nestin, indicating stem cell phenotype. The cells differentiated from the neurospheres were positive for GFAP,?-tubulin III and MBP, indicating the CD133~+ neurosphere tumor cells capable of multipotent differentiation. The average size and volume of the xenographed tumors formed by the CD133~+ cells significantly larger compared with that formed by CD133- cells. These results suggest that cells possessed stem-like cell property, i.e. GSCs.2. The expression of miR-20/106 and TIMP-2 pathway in human GSCs.Compared with GCs, the expression level of miR-20/106 was significantly increased in GSCs (P<0.05). These data were in consistent with the results from microarray hybridization. The quantitative analysis and western blot showed an overall lower expression of TIMP-2 at mRNA and protein levels in GSCs than that in GCs (P<0.05). Nevertheless, the mRNA and protein expression of MMP2 and MMP9 in GSCs were significantly higher than that in GCs (P<0.05).3. miR-20/106 promoted invasion of human GSCs.We found that GSCs invasiveness were notably higher than GCs (P<0.05). Ectopic transfection of miR-20/106 inhibitors led to significantly decreased invasion of GSCs when compared to the cells tranfected with the negative control (P<0.05).4. miR-20/106 directly regulated TIMP-2 expression.The mRNA and protein expression of TIMP-2 in these cells transfected with miR-20/106 inhibitors were significantly increased compared with the GSCs transfected with a scrambled miRNA inhibitors control (P<0.05). While these cells showed significantly lower MMP2 and MMP9 expression than the GSCs transfected with a scrambled miRNA inhibitors control (P<0.05).Transfection of GSCs with a miR-20/106 inhibitors significantly increased the luciferase activity of the reporter vectors containing the 3'UTR of a wild-type TIMP-2, whereas miR-20/106 mimics suppressed that of the cells (P<0.05). Both miR-20/106 inhibitors and mimics had no effect on the luciferase activity of the reporters with a mutant TIMP-2 3'UTR (P>0.05).Conclusions:1. The CD133~+ cells from human GBM cell line U87 and primary human GBM samples showed biological features of cancer stem cells, i.e. GSC.2. GSCs possessed significantly enhanced invasiveness compared with differentiated glioma cells (GCs).3. miR-20/106 expression was markedly increased in GSCs from U87 and primary glioma cells compared with GCs, which might contribute to the enhanced potential of invasiveness of GSCs by down-regulating at least in part the expression of TIMP-2 protein.4. TIMP-2 is down-regulated in GSCs because of miR-20/106 augmentation.