Polymorphisms in microRNA target sites modulate risk of lymphoblastic and myeloid leukemias and affect microRNA binding

Abstract

Background: MicroRNA dysregulation is a common event in leukemia. Polymorphisms in microRNA-binding sites (miRSNPs) in target genes may alter the strength of microRNA interaction with target transcripts thereby affecting protein levels. In this study we aimed at identifying miRSNPs associated with leukemia risk and assessing impact of these miRSNPs on miRNA binding to target transcripts.

Methods: We analyzed with specialized algorithms the 3' untranslated regions of 137 leukemia-associated genes and identified 111 putative miRSNPs, of which 10 were chosen for further investigation. We genotyped patients with acute myeloid leukemia (AML, n = 87), chronic myeloid leukemia (CML, n = 140), childhood acute lymphoblastic leukemia (ALL, n = 101) and healthy controls (n = 471). Association between SNPs and leukemia risk was calculated by estimating odds ratios in the multivariate logistic regression analysis. For miRSNPs that were associated with leukemia risk we performed luciferase reporter assays to examine whether they influence miRNA binding.

Results: Here we show that variant alleles of TLX1_rs2742038 and ETV6_rs1573613 were associated with increased risk of childhood ALL (OR (95% CI) = 3.97 (1.43-11.02) and 1.9 (1.16-3.11), respectively), while PML_rs9479 was associated with decreased ALL risk (OR = 0.55 (0.36-0.86). In adult myeloid leukemias we found significant associations between the variant allele of PML_rs9479 and decreased AML risk (OR = 0.61 (0.38-0.97), and between variant alleles of IRF8_ rs10514611 and ARHGAP26_rs187729 and increased CML risk (OR = 2.4 (1.12-5.15) and 1.63 (1.07-2.47), respectively). Moreover, we observed a significant trend for an increasing ALL and CML risk with the growing number of risk genotypes with OR = 13.91 (4.38-44.11) for carriers of ≥3 risk genotypes in ALL and OR = 4.9 (1.27-18.85) for carriers of 2 risk genotypes in CML. Luciferase reporter assays revealed that the C allele of ARHGAP26_rs187729 creates an illegitimate binding site for miR-18a-3p, while the A allele of PML_rs9479 enhances binding of miR-510-5p and the C allele of ETV6_rs1573613 weakens binding of miR-34c-5p and miR-449b-5p.

Conclusions: Our study implicates that microRNA-binding site polymorphisms modulate leukemia risk by interfering with the miRNA-mediated regulation. Our findings underscore the significance of variability in 3' untranslated regions in leukemia.

Figure 1

Effect of miRSNPs on miRNA binding and protein expression. A-C) Jurkat cells were transfected in triplicate with 1 µg either empty psiCheck2 vector or psiCheck2 constructs containing 3′UTRs with wild-type and variant alleles, with miRNA mimics, inhibitors or miRNA negative control (50 pmol/well). 24 h post transfection luciferase activity was measured. Data show relative Renilla luciferase levels normalized to firefly luciferase and corrected for the effect of miRNA mimics on the empty psiCheck2 vector. Values for the miRNA negative control were set as 100%. All transfections were done three times. * p < 0.05, ** p < 0.01, *** p < 0.001. Sequence alignments of D) miR-18a-3p with the ARHGAP26 3′UTR, E) miR-34c-5p and miR-449b-5p with the ETV6 3′UTR and F) miR-510-5p and miR-589-3p with the PML 3′UTR. MiRNA seed sequences are underlined. Allelic variants in each 3′UTR are underlined and in bold. For miR-510-5p its seed sequence repeated twice is shown (one in bold, the other underlined) instead of the entire mature miRNA sequence to show its complementarity with two adjacent miR-510 binding sites in the PML 3′UTR.