Page 165 - Molecular features of low-grade developmental brain tumours

MIR-519D & MIR-4758 CAN IDENTIFY GG FROM DNT AND ASTROCYTOMAS
miRNA target expression in GG
In order to identify potential targets of miR-519d and miR-4758, 5 different databases were utilised. Targets were considered relevant if found in at least 3 of the 5 databases. Based on these target prediction tools, AKT3, CDKN1A (P21), JAK1, PTEN, ERBB3, ERBB4, RB1 and TIMP2 were identified as potential targets of miR-519d. Both clinical and experimental studies have shown the potential contribution of miR-519d dysregulation in hepatocellular carcinoma, breast cancer and cervical cancer 37-39. AKT3, CDKN1A and PTEN have all been shown to be downregulated by miR-519d, potentially explaining the oncogenic features of this miRNA 40,41. Based on target prediction tools the relatively unknown miRNA, miR-4758 was predicted to target CDK2 and CDKN1B (P27), raising the possibility that miR-4758 also has oncogenic properties. Evaluation of the mRNA expression of miR-519d and miR-4758 targets (Fig. 3) showed a downregulation of ERBB3 (p=0.0005), AKT3 (p=0.0015), PI3KCA (p=0.0015), RB1 (p=0.0009), PTEN (p=0.0041), TP53 (p=0.023) and CDKN1B (p=0.0065) in GG (n=14, except for ERBB4 where n=13) compared to control cortex (n=7; Mann-Withney U test). Furthermore, targets ERBB4 (p=0.032) CDKN1A (p=0.048) and JAK1 (p=0.0124) and were found to be upregulated in GG compared to control cortex. The expression of TIMP2 (p=0.68), CDK4 (p=0.39), CDK2 (0.91) and MDM2 (p=0.63) did not change.
miRNA target expression in cell culture
Given the lack of GG cell lines, we used the human pediatric low grade astrocytoma cell line Res259 (LGG2) obtained from a pediatric AII 42 to investigate the effects of miR-519d and miR-4758 upregulation. After transfection with miRNA mimic, overexpression of the specific miRNA was observed under basal condition (data not shown). Quantitative real-time PCR analysis was performed to evaluate the effects of miR-519d and miR-4758 transfection on mRNA expression of a subset of targets related to the PI3K/AKT pathway, involved in the regulation of cell cycle progression43. CDKN1A was downregulated after miR- 519d transfection, whereas transfection with miR-4758 did not affect CDKN1A expression compared to control (Fig. 4A). Co-transfection of miR-519d with miR-4758 rescued the effect of miR-519d transfection. The expression of miR-4758 target CDKN1B was not affected by transfection with miR-4758 or miR-519d alone (Fig. 4B). However, after co-transfection with miR-4758 and miR-519d CDKN1B was upregulated compared to control. Furthermore, PTEN mRNA expression did not change after transfection with either one of the miRNAs (Fig. 4C).
Effects of miR-519d and miR-4758 modulation on the cell cycle
We further evaluated the role of miR-519d and miR-4758 on cell cycle progression using flow cytometry analysis in Res259/LGG2. Overexpression of miR-519d showed a decreased G1 phase (13%, p<0.01) whereas, transfection with miR-4758 resulted in an increased G2/M phase. Overexpression of miR-519d, but not miR-4758, increased the S phase; miR-4758 mimic co-transfected with miR-519d mimic was able to counteract the effect of miR-519d alone (Fig. 4D).
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