B Cell Receptor Stimulation of CLL Cells Leads to Upregulation of IRF4 Proteinexpression Influenced by SNP Expression,

As previously published by Di Bernado et al. (2008) the single nucleotide polymorphism (SNP) RS872071 located in the 3'UTR of IRF4 (Interleukin Regulatory Factor 4) has influence on the risk for developing chronic lymphocytic leukemia (CLL). IRF4 is a key player in the development of B lymphocytes and multiple myelomas. The SNP is either expressed as Adenine (A) or Guanine (G). Expression of G/G increases the risk to develop CLL. MicroRNAs (miRNAs) adhere posttranscriptional to the 3'untranslated region (UTR) of mRNAs and can influence the expression of mRNA and proteins. The SNP lies within the 3'UTR of IRF4 and offers some putative binding sites for miRNAs. The aim of this project is to proof that this SNP has influence on the binding behavior of miRNAs, which are influencing the IRF4 expression.

Methods and results: To identify miRNAs binding to this region we cloned the neighboring region of the SNP into a luciferase expressing vector and generated the SNP by mutagenesis. In luciferase assays 15 miRNAs were checked for differences in binding affinity dependent on SNP expression. Three of them showed significant SNP dependent binding behavior. In all cases expression of SNP G leads to reduced luciferase expression, indicating suppression of IRF4. To elucidate the consequences on SNP expression on cellular level in CLL we collected DNA derived from B cells of CLL patients (n=104) and sequenced the SNP region. All together our pool of patients expressed 25% A/A, 31% A/G and 44% G/G. MRNA and protein expression of IRF4 was considered SNP-dependently and compared to healthy B cells. On mRNA and protein level CLL cells have a significant higher IRF4 expression compared to healthy B cells. SNP-dependent comparison between CLL cells on mRNA-level shows a tendency of less IRF4 expression in B cells of patients expressing the G/G SNP compared to patients expressing A/A SNP. However on protein level this tendency was not detected. As IRF4 is known as a key regulator for extracellular stimuli, we focused on SNP dependent IRF4 regulation after different stimuli. Whereas CD40 and IL-4 stimulation did not show SNP dependent regulation, stimulation of the B cell receptor (BCR) leads to a higher IRF4 induction in patients carrying A/A (n=6) compared to patients carrying G/G (n=5) and A/G (n=8) (p<0.05).

For the first time connections between the SNP RS872071 and molecular mechanisms explaining his influence on the pathogenesis on CLL were drawn. Dependent on expressed SNP miRNAs bind with different affinities to the 3'UTR. In further experiments the connection between IgM stimulation and miRNA expression has to be tightened.

No relevant conflicts of interest to declare.