Abstract
BackgroundGlioblastoma multiforme (GBM) is the most aggressive type of glioma and carries the poorest chances of survival. There is therefore an urgent need to understand the mechanisms of glioma tumorigenesis and develop or improve therapeutics. The aim of this study was to assess the possible prognostic value of cyclin-dependent kinase 6 (CDK6) and the effects of microRNA-495 (miR-495) manipulation on CDK6 expression and cell survival in glioma cells.MethodsAnalyses of clinical specimens from GBM patients were used. Expression of CDK6 was analyzed by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. Expression of CDK6 was also analyzed after over-expression of miR-495 in T98 cells; both cell proliferation and RB phosphorylation were examined. Cell proliferation, cell cycle distribution, and RB phosphorylation were also examined after knockdown of CDK6 in U87-MG and T98 cells.ResultsAnalyses of clinical specimens from GBM patients identified that CDK6 is significantly expressed in gliomas. CDK6 antigen expression was higher in tumor cores and margins than in adjacent normal brain tissues, and higher levels of CDK6 expression in the tumor margin correlated with decreased survival. Over-expression of miR-495 in T98 cells downregulated the expression of CDK6 and inhibited retinoblastoma phosphorylation, and knockdown of CDK6 in U87-MG and T98 cells by siRNAs resulted in cell cycle arrest at the G1/S transition and inhibition of cell proliferation.ConclusionsThis study revealed miR-495 is down-regulated in glioma tissues. Furthermore, miR-495 regulated CDK6 expression and involved in glioma cell growth inhibition, which indicated the possible role of miR-495 in tumor progression.
Highlights
Glioblastoma multiforme (GBM) is the most aggressive type of glioma and carries the poorest chances of survival
Correlation of cyclin-dependent kinase 6 (CDK6) expression with survival rates Kaplan-Meier survival plots showed a relationship between CDK6 expression and survival rates in GBM patients
Cell cycle analyses by flow cytometry showed that T98 and U87-MG cells transfected with siCDK6 had a higher percentage of cells in G0/G1 phase than negative control cells (Figure 3c)
Summary
Glioblastoma multiforme (GBM) is the most aggressive type of glioma and carries the poorest chances of survival. The aim of this study was to assess the possible prognostic value of cyclin-dependent kinase 6 (CDK6) and the effects of microRNA-495 (miR-495) manipulation on CDK6 expression and cell survival in glioma cells. The cell cycle regulator enzyme cyclin-dependent kinase 6 (CDK6) is over-expressed in GBM [4,5], not all the over-expression can be explained by genomic amplification, suggesting that other gene regulatory mechanisms might be involved [5]. MiRs can destabilize and mediate the degradation of RNA transcripts [9] In addition to their role in normal development, miRs are associated with carcinogenesis [10,11]. Some miRs act as tumor suppressors: miR-34 functions as a p53-dependent tumor suppressor in neuroblastoma [13], and miR-124 inhibits CDK6 and reduces tumor proliferation in medulloblastoma [14]
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