Abstract 1914: NF-κB Regulates the HSP70 Locus in Late IPC by Transcriptional and Post-transcriptional Mechanisms Involving miRNA
Introduction: It is has been demonstrated that the heat shock protein 70 (Hsp70) locus is required for late IPC. However, this locus consists of closely related genes, Hsp70.1 and Hsp70.3, that have been assumed to be redundantly protective.
Results: We show that the Hsp70.3 isoform is induced after ischemic preconditioning (IPC) and contributes significantly to the late phase of IPC cardioprotection. However, the Hsp70.1 isoform, a nearly identical gene with conserved promoter organization, does not contribute to IPC induced cardioprotection and is pro-cell death after ischemia/reperfusion injury. Both NF-κB and Hsp70.3 are required for development of cardioprotection against MI after late IPC. Interestingly, blockade of NF-κB only partially (50%) reduces IPC-induced expression of Hsp70.3 mRNA, while it completely blocks the increase of Hsp70.3 protein. We hypothesized that, in addition to regulating Hsp70.3 mRNA expression changes post-IPC, NF-κB also regulates expression of microRNAs that in turn impose post-transcriptional regulation on Hsp70.3 expression. To test this, we performed comparative miRNA expression arrays in wild-type and NF-κB dominant-negative mice after IPC, with the goal of delineating miRNAs that meet two criteria, they are;
downregulated in an NF-κB-dependent manner post-IPC, and
predicted to target Hsp70.3. The miRNAs delineated by the array were validated by quantitative reverse transcriptase-PCR.
A single miRNA was identified by this screen and was then tested for its functional ability to target Hsp70.3. The miRNA was transfected into primary murine fibroblasts isolated from Hsp70.1 KO mice to validate targeting and regulation of Hsp70.3 protein levels.
Discussion: This work is the first to show NF-κB-dependent transcriptional and post-transcriptional (via miRNA) co-regulation of a cardioprotective gene after late IPC. Understanding miRNA regulatory networks and how they integrate with transcriptional networks will enhance our mechanistic understanding of cardioprotection and may have therapeutic application, particularly in the use of RNAi and miRNA for regulatory therapy.