A Targeted Resequencing Study Allows the Detection of a Common Polymorphism At Mirna-223 Binding Site in 3′Untranslated Region Which Deregulates HSP90B1 Expression in Chronic Lymphocytic Leukemia

Abstract 1776

Array-based sequence capture (Roche NimbleGen) followed by next-generation sequencing (Roche GS FLX Titanium sequencing platform) was used to analyze genetic variations in 93 genes relevant in CLL and two chromosomal regions: 13q14.3 and 17p13.1. CD19+ cells from 4 patients with CLL and 4 patients with other hematological malignancies (used as controls) were studied. A custom-made data analysis pipeline was used to annotate detected variants, including known single-nucleotide polymorphisms (SNPs), amino acid consequences, genomic location and miRNA binding sites. The enrichment assay followed by NGS allowed the detection of over 1600 variations/sample (median 1721, range 1618–1823). All putative variants were first compared with published single nucleotide polymorphism (SNP) data (dbSNP build 130) and most of the variants detected were identified as known SNPs. Overall, 10% of variants detected in each sample were variations not previously described. Interestingly, a 4bp insertion/deletion polymorphism (rs2307842) in the 3′UTR of HSP90B1, target site for miR-223, was detected. There is an increasing evidence suggesting that SNPs in the 3′UTR targeted by miRNAs (known as miRSNPs) are associated with diseases by affecting gene expression. We hypothesized that this ‘GACT’ deletion disrupts the binding site for miR-223 thereby increasing the translation of HSP90B1 and we confirmed that miR-223 regulates HSP90B1 expression by 3′UTR reporter assays. The relative luciferase activity of the construct with wild-type 3′UTR (WT-3′UTR) was significantly repressed by 31% following miR-223 transfection (p<0.05). However, the presence of rs2307842 polymorphism in 3′UTR of HSP90B1 (VAR-3′UTR) abolished this suppression, suggesting that miR-223 directly binds to this site. We also validated HSP90B1 as a target gene of miR223 by transfecting MM1S and H929 cell lines with miR-223/NC mimics and then measuring HSP90B1 expression by semi-quantitative PCR and Western blot. Exogenous expression of miR-223 downregulated the expression levels of HSP90B1 in H929 cell line (WT-3′UTR) in both mRNA (p<0.05) and protein levels. By contrast, HSP90B1 expression was not modified in MM1S cell line (VAR-3′UTR). To evaluate the clinical impact of HSP90B1 3′UTR polymorphism, we expanded the study to 109 additional patients with CLL and 32 healthy controls. Sequencing of the HSP90B1 3′UTR region was performed by pyrosequencing (PyroMark Q24 system, Qiagen). The rs2307842 was detected in 27/109 (25%) patients and 8/32 (25%) healthy controls, as expected. Overall, we did not find any significant relationship between rs2307842 and clinical characteristics of CLL patients. To gain insight into its influence on gene expression, we measured HSP90B1 mRNA levels in paired samples (tumoral and normal) from CLL patients with rs2307842 (VAR-CLLs, n=6) and wild-type (WT-CLLs, n=12). PCR results showed that B lymphocytes (tumoral fraction) from VAR-CLLs have a higher expression of HSP90B1 than B lymphocytes from WT-CLLs (P=0.002) and also from the normal cells of the same patients (VAR-CLLs) (P=0.011). However, in WT-CLLs, no changes in mRNA expression were observed between tumor and normal fractions, being HSP90B1 mRNA levels similar to the normal fraction of VAR-CLLs. Thus, rs2307842 determined HSP90B1 overexpression only in the tumor fraction of the CLL patients with the polymorphism.

Downregulation of miR-223 has prognostic significance in CLL. However, there is no evidence of the pathogenetic mechanism of this miRNA in CLL patients, and no target has been proposed or validated for miR-223 in CLL until date. Thus, this work provides novel information about how the downregulation of miR-223 can be determining the poor outcome of CLL patients, maybe through upregulation of HSP90B1 expression.