Study of Lcn2 in inflammation and characterization of its RNA aptamer
Lipocalin-2 (Lcn2), a member of the lipocalin superfamily, has been implicated in diverse physiological and pathological processes, such as apoptosis, cell differentiation, inflammation, iron metabolism and wound injury healing. However, most of these reports are of correlations and no cause-effect relationship has been established for Lcn2's role in inflammation and wound healing. Using Lcn2 gene knockout mice (Lcn2-/-), we investigated the role of Lcn2 in both lipopolysaccharide (LPS)-induced acute lung inflammation response and dextran sulfate sodium (DSS)-induced colitis. Under all the treatment conditions, no significant differences were observed in proinflammatory cytokines expression level between the LCN2-/- and wild-type mice for the lung inflammation model. For both of animal models, the histological studies and the observed disease severity also indicated no significant difference between these two types of mice. We conclude from the results of this study that Lcn2 has no causal role in the induction of inflammation nor does it play a protective role against inflammation.
In addition to the functional/mechanism studies, Lcn2 has been re-evaluated as a standard biomarker for many diseases. To develop a new detection tool for Lcn2, in the current study we selected and characterized a mouse Lcn2 (mLcn2) RNA aptamer. Binding affinity analysis demonstrated this RNA aptamer has a dissociation constant for mLcn2 of 0.34 ± 0.07 μM, but does not bind the human and chicken forms of Lcn2. RNA foot printing and mutational assay indicated that the nucleic acids to protein contact regions are mainly located on the loops of the aptamer, which was predicted to fold in a 3-way junction secondary structure. Further analysis from site mutagenesis of mLcn2 revealed that the aptamer-protein interaction involves the amino acids in the pocket of mLcn2 that normally bind its native ligand, iron-siderophore, but does not involve the mLcn2 surface polar amino acids. Application of the mLcn2 aptamer as a detection probe in a homogenous assay was also demonstrated by using micro-cantilever system.