Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Aug;5(3):183-91.
doi: 10.1007/s12079-011-0139-x. Epub 2011 Jun 3.

MicroRNAs 130a/b are regulated by BCR-ABL and downregulate expression of CCN3 in CML

Sukanya Suresh  1 , Lynn McCallum, Wanhua Lu, Noureddine Lazar, Bernard Perbal, Alexandra E Irvine
Affiliations

MicroRNAs 130a/b are regulated by BCR-ABL and downregulate expression of CCN3 in CML

Sukanya Suresh et al. J Cell Commun Signal. 2011 Aug.

Abstract

Chronic Myeloid Leukaemia (CML) is a myeloproliferative disorder characterized by the expression of the oncoprotein, Bcr-Abl kinase. CCN3 normally functions as a negative growth regulator, but it is downregulated in CML, the mechanism of which is not known. MicroRNAs (miRNAs) are small non-coding RNAs, which negatively regulate protein translation by binding to the complimentary sequences of the 3' UTR of messenger RNAs. Deregulated miRNA expression has emerged as a hallmark of cancer. In CML, BCR-ABL upregulates oncogenic miRNAs and downregulates tumour suppressor miRNAs favouring leukaemic transformation. We report here that the downregulation of CCN3 in CML is mediated by BCR-ABL dependent miRNAs. Using the CML cell line K562, we profiled miRNAs, which are BCR-ABL dependent by transfecting K562 cells with anti-BCR-ABL siRNA. MiRNA expression levels were quantified using the Taqman Low Density miRNA array platform. From the miRNA target prediction databases we identified miRNAs that could potentially bind to CCN3 mRNA and reduce expression. Of these, miR-130a, miR-130b, miR-148a, miR-212 and miR-425-5p were significantly reduced on BCR-ABL knockdown, with both miR-130a and miR-130b decreasing the most within 24 h of siRNA treatment. Transfection of mature sequences of miR-130a and miR-130b individually into BCR-ABL negative HL60 cells resulted in a decrease of both CCN3 mRNA and protein. The reduction in CCN3 was greatest with overexpression of miR-130a whereas miR-130b overexpression resulted only in marginal repression of CCN3. This study shows that miRNAs modulate CCN3 expression. Deregulated miRNA expression initiated by BCR-ABL may be one mechanism of downregulating CCN3 whereby leukaemic cells evade negative growth regulation.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
BCR-ABL silencing increases CCN3 expression. siRNA directed to target BCR-ABL was used to investigate CCN3 expression levels. K562 cells transfected with siRNA targeting BCR-ABL, a scrambled sequence and untransfected control cells were compared at 24 h and 72 h post transfection. a Real time PCR was performed to quantify BCR-ABL transcript levels. A significant reduction in BCR-ABL was observed within 24 h of siRNA treatment. b RQ-PCR was performed to detect levels of CCN3 expression as a result of siRNA targeting BCR-ABL. BCR-ABL reduction corresponded with an increase in CCN3. cBox plots indicating the difference in CCN3 gene expression in each set of samples. Data represent normalized Ct values; higher the Ct value, lower the gene expression. d Western blotting was performed on protein lysates to detect expression of Bcr-Abl (i), Ccn3 (ii) and β-actin. e Optical densitometry was performed for the blots in (c) and the data was graphically formatted to compare levels of Bcr-Abl and Ccn3 protein expression normalized to β-actin. Data represent the mean of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001
Fig. 2
Fig. 2
The expression of miRNAs predicted to target CCN3 decreases with BCR-ABL reduction. Analysis of BCR-ABL dependent miRNA expression of K562 cells was carried out using TLDA miRNA cards. The miRNA expression of K562 cells transfected with siRNA directed against BCR-ABL was compared with the control K562 cells at 24 h and 72 h post transfection. Data represents the fold reduction observed in the expression of 5 miRNAs predicted to target CCN3 with each BCR-ABL silencing experiment (n = 3), *, p < 0.05; ***, p < 0.001. The expression of RNU48 was used as the endogenous control. Light bars represent 24 h and dark bars represent 72 h time points
Fig. 3
Fig. 3
BCR-ABL associated miRNAs change expression with respect to BCR-ABL reduction. The expression of two of the BCR-ABL associated miRNAs was identified using the TLDA platform. a The miR-17-92 cluster upregulated by BCR-ABL showed significant reduction at both 24 h and 72 h post BCR-ABL siRNA transfection in K562 cells. b The tumour suppressor miR-328 was significantly increased at 72 h of BCR-ABL silencing. Data represent the mean fold reductions observed in three independent experiments. *, p < 0.05;; ***, p < 0.001. Light bars represent 24 h and dark bars represent 72 h time points
Fig. 4
Fig. 4
Imatinib treatment of K562 cells reduces miR-130a and miR-130b expression. K562 cells were treated with 1 μM imatinib for 24, 48 and 72 h and the expression levels of miR-130a and miR-130b were compared with untreated K562 cells. Quantitative PCR for individual miRNAs were performed and expression was normalized to the endogenous control RNU48. (a) Western blotting showing the decrease in Bcr-Abl expression (i) and the corresponding increase in Ccn3 (ii) in K562 cells with 24, 48 and 72 h of imatinib treatment (b) Expression levels of miR-130a and (c) miR-130b following 24, 48 and 72 h of imatinib treatment of K562 cells. Data represent the mean fold reduction in miR-130a and miR-130b expression relative to the untreated K562 cells (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001
Fig. 5
Fig. 5
Overexpression of miR-130a and miR-130b reduces CCN3 levels. (a) Real time PCR analysis of CCN3 copy number in K562 cells and BCR-ABL negative HL60 cells. (b) Amaxa transfection of miRNA mimics of miR-130a showed a significant increase in miR-130a transcripts at 24 h and 48 h following transfection, (c) transfection of miR-130b miRNA mimics show increased expression of miR-130b in HL60 cells at both 24 h and 48 h (d) CCN3 mRNA levels decrease with miR-130a and miR-130b overexpression at 24 h and 48 h post transfection of the miRNA mimics (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001. (e) Corresponding protein level determination, miR-130a overexpression decreases Ccn3 at 48 h where as miR-130b transfection shows a slight decrease at Ccn3 protein levels. (f) Corresponding optical density readings for the blots in (e) comparing the levels of Ccn3 protein normalized to β-actin
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bhatia R, Holtz M, Niu N, Gray R, Snyder DS, Sawyers CL, Arber DA, Slovak ML, Forman SJ. Persistence of malignant hematopoietic progenitors in chronic myelogenous leukemia patients in complete cytogenetic remission following imatinib mesylate treatment. Blood. 2003;101:4701–4707. doi: 10.1182/blood-2002-09-2780. - DOI - PubMed
    1. Bueno MJ, Pérez de Castro I, Gómez de Cedrón M, Santos J, Calin GA, Cigudosa JC, Croce CM, Fernández-Piqueras J, Malumbres M. Genetic and epigenetic silencing of microRNA-203 enhances ABL1 and BCR-ABL1 oncogene expression. Cancer Cell. 2008;13:496–506. doi: 10.1016/j.ccr.2008.04.018. - DOI - PubMed
    1. Deininger MW, Goldman JM, Melo JV. The molecular biology of chronic myeloid leukemia. Blood. 2000;10:3343–3356. - PubMed
    1. Druker BJ, Lydon NB. Lessons learned from the development of an abl tyrosine kinase inhibitor for chronic myelogenous leukemia. J Clin Invest. 2000;105:3–7. doi: 10.1172/JCI9083. - DOI - PMC - PubMed
    1. Duisters RF, Tijsen AJ, Schroen B, Leenders JJ, Lentink V, Made I, Herias V, Leeuwen RE, Schellings MW, Barenbrug P, Maessen JG, Heymans S, Pinto YM, Creemers EE. miR-133 and miR-30 regulate connective tissue growth factor: implications for a role of microRNAs in myocardial matrix remodeling. Circ Res. 2009;104:170–178. doi: 10.1161/CIRCRESAHA.108.182535. - DOI - PubMed