MicroRNAs regulating CCN3 expression in Chronic Myeloid Leukaemia


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Sukanya Suresh1, Lynn McCallum1, Sandra Irvine1
1Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, United Kingdom

Background

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.

Method

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.

Results

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).

Conclusion

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.