A 40-Microrna Signature Characterizes JAK2V617F-Negative Essential Thrombocythemia: Role Of Mir-221 and Mir-203 In The Regulation Of The JAK-STAT Pathway

Abstract

BackgroundMicroRNAs (miRNAs) act as negative regulators of expression of genes that participate in cellular proliferation, apoptosis and/or carcinogenesis. MiRNAs have been shown to play a role in both solid and hematological tumors. Interestingly, several miRNA profiles have been differentially related to the mutational status of multiple tumors. A mutation in the JAK2 kinase (V617F) has been associated with essential thrombocythemia (ET) in around 40–50% of cases. However, the biological basis of ET in patients lacking the JAK2V617F mutation is still unknown. miRNAs may be crucial to the development of ET in these patients, yet their role in JAK2V617F-negative ET remains to be elucidated. AimTo characterize the expression pattern of miRNAs involved in JAK2V617F-negative ET and to identify the targets of these miRNAs. MethodsTotal RNA was extracted from isolated platelets from 19 ET patients (10 JAK2V617F-positive and 9 JAK2V617F-negative) and 10 healthy controls. The expression of 670 mature miRNAs was determined by TaqMan Human MicroRNA Arrays v2.0 (Applied Biosystems) in an ABI 7900 HT real time PCR system. miRNA expression data was analyzed by the 2-ΔΔCt method, using RNU48 as endogenous control. Statistical analyses were performed with TiGR MultiExperiment Viewer and R software v 2.13.To identify molecular pathways potentially altered by the expression of multiple miRNAs, we used Diana-mirPath, which performs an enrichment analysis of multiple miRNA target genes, comparing each set of miRNA targets to all known KEGG pathways. The mRNA expression of identified putative targets was analyzed by TaqMan gene expression assays (Applied Biosystems) and correlated with miRNA expression in order to select candidate genes for further target validation by Renilla-Luciferase assay and Western Blot. For Renilla-Luciferase assay, 100nM of the pre-miRNAs of interest or pre-miR negative control were transfected in K562 together with 1μM of modified psicheck2 vector containing the 3'UTR region to be validated. Renilla luciferase levels were measured at 24h post-tranfection. The miRNAs identified in the Renilla-Luciferase assay were further confirmed by Western Blot. 100nM of pre-miR/pre-miR negative control were transfected in the HL60 cell line and protein levels were measured at 24h post-transfection. Anti-SOCS1 (ab119954) and Anti-SOCS3 (ab62584) primary antibody (abcam) were used. ResultsHierarchical cluster analysis showed two well-separated clusters between patients and controls, indicating that ET platelets had a characteristic miRNA signature (p<0.0001). ET patients harbored a distinctive signature of 101 miRNAs, 99 of which were downregulated. Furthermore, a 40-miRNA signature was found to be differentially expressed between JAK2V617F-positive and JAK2V617F-negative ET patients. Diana-mirPath analysis showed that 8 of these 40 miRNAs had putative targets in the JAK-STAT pathway. The mRNA expression analysis of these genes showed an inverse correlation between the expression of three miRNAs and their target genes. An inverse correlation was found between SOCS1 and miR-221 (r2 = -0.719, p=0.001), SOCS3 and miR-221 (r2 = -0.644, p=0.005), SOCS3 and miR-203 (r2 = -0.447, p=0.072) and PTPN11 and miR-23a (r2 = -0.494, p=0.044). All three miRNAs were upregulated in JAK2V617F-negative ET patients in comparison with JAK2V617F-positive ET patients. The Renilla-Luciferase assay validated SOCS1 as a target of miR-221 (28.9% Renilla luciferase protein reduction, p=0.002) and SOCS3 as a target of miR-203 (19.6% Renilla luciferase protein reduction, p=0.04). No significant modifications were observed for PTPN11. SOCS1 and SOCS3 were further validated by Western Blot, with significant reduction of protein levels of SOCS1 (16%) and SOCS3 (19%) after increasing the levels of miR-221 and miR-203 respectivelly. ConclusionA 40-miRNA signature is differentially expressed in JAK2V617F-negative and JAK2V617F-positive ET patients. Two of these miRNAs, miR-221 and miR-203, target SOCS1 and SOCS3, which are involved in the JAK-STAT pathway. Further investigation is warranted to shed light on the role of these miRNAs in the development of JAK2V617F-negative ET. AcknowledgmentsThis study was supported in part by grants EC10-136, FISPI10/01807, RD09/0076/00036, RD12/0036/0010, 2009SGR929 and AECC Catalunya 2011 Disclosures:No relevant conflicts of interest to declare.