MicroRNAs regulating CCN3 expression in Chronic Myeloid Leukaemia
Session type: Poster / e-Poster / Silent Theatre session
MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate protein expression. Chronic Myeloid Leukaemia (CML) is characterized by the Bcr-Abl oncogene which has been shown to deregulate miRNA expression, favouring disease progression. Previously we found that CCN3, a tumour supressor in CML, is downregulated by Bcr-Abl. This study investigates Bcr-Abl regulation of miRNA(s) repressing CCN3 protein expression in the human CML cell line, K562.
K562 cells were transfected with anti-Bcr-Abl siRNA (0.5 ?g) using Amaxa nucleofection. Bcr-Abl reduced by 3.8 fold at 24h and 3.3 fold at 72h following siRNA treatment. A corresponding 3.4 fold increase in CCN3 was observed at 24h (p < 0.0005) and CCN3 levels were maintained at 72h (3.7 fold, p < 0.001). The miRNA expression in K562 cells before and after Bcr-Abl silencing (24h and 72h) was assayed using TaqMan® Low Density Human MicroRNA Array cards. Results were analyzed by SDS Relative Quantification (RQ) Manager using 2-DDCT method.
Using Mirgen target interface, 14 miRNAs were found to target CCN3 mRNA of which mir-130b, mir-130a, mir-148a and mir-425-5p reduced more than 1.5-fold within 24 hrs of Bcr-Abl knockdown (n=3). Mir-130b with 3.4 fold reduction (p < 0.05) at 24h, and 3.14 fold reduction at 72h (p < 0.05) was selected to confirm its effect on CCN3 expression. Pre-miRNA precursor of mir-130b (30 nm) and scrambled miRNA were transfected into the human acute myeloid leukaemia cell line, HL60 (Bcr-Abl negative) and CCN3 mRNA levels were determined. Transfection of mir-130b precursor resulted in 1.8 fold reduction in CCN3 mRNA levels at 24h (p < 0.01) and 2.02 fold reduction (p < 0.05) at 48h (n=3).
This study shows that Bcr-Abl induced upregulation of miRNAs including mir-130b, could contribute to reduced CCN3 expression in CML cells. Further validation of these miRNAs may provide novel therapeutic strategies to target CML.