SUN-078 ADAM12 Is a Direct Target of mir29a: Implications for Impaired Post-Ischemic Adaptation in Diabetes

We previously identified ADAM12 as a gene that is upregulated in ischemic hind limb of mice and play a critical role in perfusion recovery following induction of experimental peripheral arterial disease. Additionally, we showed that miR29a is a microRNA that regulates ADAM12 expression in ischemic endothelial cells and ischemic mouse hind limbs. Moreover, we also show that miR29a regulation of ADAM12 in ischemia is impaired in vitro and in vivo in the setting of high glucose or diabetes. The effects of miR29a could be due to direct effects on ADAM12 mRNA or indirectly through secondary effects on the transcriptome. To begin to understand how miR29a regulation of ADAM12 is impaired in high glucose, we explored whether ADAM12 is a direct target of miR29a. We performed RNA immunoprecipitation (RIP) based anti-miR competitive assay. miRNA negatively regulate gene expression through recruitment of the RNA induced silencing complexes (RISK) which contains the AGO2 protein bound to miRNAs and their target mRNAs. Anti-miR are synthetic oligonucleotide that binds specific miRNAs and can selectively displace the mRNA’s binding to its target mRNA transcripts. The RIP competition assay involves titrating different concentrations of an anti-miR into cell or tissue lysates followed by immunoprecipitation (IP) of AGO2 and analysis of the mRNAs within the AGO2 protein. Addition of anti-miRs results in displacement of the mRNA transcripts that are direct targets of the miRNA of interest. Therefore, anti-miR dose dependent reduction in the amount of an mRNA transcript associated with IP’ed AGO2 strongly suggest the mRNA is a direct target of the miRNA. Human Umbilical Vein Endothelial Cells (HUVEC) were lysed, pre-cleared with protein A/G PLUS-Agarose, then incubated with anti-miR29a or control anti-miR at 0nM, 4nM, 40nM and 400 nM,. Each lysate was IP with AGO2 antibody plus protein A/G PLUS-Agarose. Total RNA was purified from the IP’ed complex, reverse transcribed to cDNA for qRT-PCR. Samples treated with anti-miR29a showed a dose dependent elimination of miR29a from the AGO-2 IP complex (1, 0.87, 0.68, 0.44), while samples treated with control anti-miR did not show a dose dependent change (1, 0.84, 0.81, 0.87). We also found a dose dependent elimination of ADAM12 from the AGO-2 IP complex (1, 0.89, 0.80, 0.68) in samples treated with anti-miR29a while treatment with control anti-miR showed no significant change in ADAM12 mRNA associated with the complex (1, 0.97, 1.03, 1.00). Lastly, treatment with anti-miR29a did not alter levels of a control gene not regulated by miR29a within the IP’ed complex. In conclusion our result strongly suggest ADAM12 is a direct target of miR29a and provides a key foundation for future studies to understand how diabetes contributes to impaired miR29a regulation in ischemia.