Differential expression of miR16 in glioblastoma and glioblastoma stem cells: their correlation with proliferation, differentiation, metastasis and prognosis

Abstract

The function of miR16 in multiforme glioblastoma multiforme (GBM) and its stem cells (GSCs) remains elusive. To this end, we investigated the patterns of miR16 expression in these cells and their correlation with malignant behaviors and clinical outcomes. The levels of miR16 and its targeted genes in tumor tissue of GBM and GBM SGH44, U87, U251 cells as well as their stem cell counterparts were measured by qRT–PCR or western blot or immunohistochemistry. Luciferase reporter assay was used to confirm the binding of miR16 to 3′-UTR of its target genes. The effects of miR16 on malignant behaviors were investigated, including tumor cell viability, soft-agar colony formation, GSCs Matrigel colony forming and migration and invasion as well as nude mice xenograft model. Differentially expression patterns of miR16 in glioblastoma cells and GSCs cells were found in this study. Changes of miR16 targeted genes, Bcl2 (B cell lymphoma 2), CDK6 (Cyclin-dependent kinase 6), CCND1 (cyclin D1), CCNE1 (cyclin E1) and SOX5 were confirmed in glioblastoma cell lines and tissue specimens. In vitro and in vivo studies showed that tumor cell proliferation was inhibited by miR16 mimic, but enhanced by miR16 inhibitor. The expression level of miR16 positively correlates with GSCs differentiation, but negatively with the abilities of migration, motility, invasion and colony formation in glioblastoma cells. The inhibitory effects of miR16 on its target genes were also found in nude mice xenograft model. Our findings revealed that the miR16 functions as a tumor suppressor in GSCs and its association with prognosis in GBM.

MiR‐16‐5p is frequently down‐regulated in astrocytic gliomas and modulates glioma cell proliferation, apoptosis and response to cytotoxic therapy

“…In TCGA data sets, we also addressed the question whether miR‐16‐5p expression is associated with overall survival of glioma patients. Expression of miR‐16‐5p was associated with overall survival in an institutional cohort of 132 glioblastoma patients . However, we found no prognostic role of high vs .…”

“…Following transient transfection of miR‐16‐5p precursor molecules into different glioma cell lines, we found reduced expression of each of these genes at the mRNA and/or protein levels. Previous studies had already shown that miR‐16‐5p directly binds the 3′‐UTR of CDK6 , CCND3 , CCNE1 , CDC25A and BCL2 [13,24–26,29,39,40,51,55,56]. Using 3′‐UTR luciferase reporter assays we confirmed its specific binding to the 3′‐UTR of WEE1 and CHEK1 .…”

“…In contrast, studies on glioma cell lines support a tumour suppressive role of miR‐16‐5p by demonstrating inhibitory effects of this miRNA on glioma cell proliferation, migration, invasion and epithelial‐to‐mesenchymal transition, as well as stimulatory effects on apoptosis . Recent data additionally reported that miR‐16‐5p inhibits glioma stem cell clonogenicity and xenograft growth . In line, we found that miR‐16‐5p inhibited proliferation of three glioma cell lines (A172, TP365MG, T98G) and increased caspase‐3/7 activity in two of these (A172, TP365MG).…”

“…miRNA target prediction showed that miR‐16‐5p has binding sites in the 3′‐UTR of several genes that are involved in cell cycle progression and apoptosis. Most of these miR‐16‐5p targets have been validated by 3′‐UTR luciferase assays in previous studies, including CDK6, CCND3, CCNE1 , CDC25A , WEE1, CHEK1 and BCL2 . To investigate whether these targets are regulated by miR‐16‐5p in glioma cells, we transiently transfected A172 glioma cells with pre‐miR‐16‐5p or pre‐miR‐NC.…”

“…At the same time, miR‐16‐5p down‐regulation may facilitate glioma cell proliferation due to enhanced G1/S phase transition caused by up‐regulation of e.g. CDK6 and CDC25A .…”