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Abstract: TH-PO780

Identification of Disease-Relevant MicroRNA-Target Gene Interactions

Session Information

Category: Glomerular Diseases

  • 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix

Authors

  • O'Connor, Christopher Lund, University of Michigan, Ann Arbor, Michigan, United States
  • Shi, Jian, University of Michigan, Ann Arbor, Michigan, United States
  • Zhang, Huanqing, University of Michigan, Ann Arbor, Michigan, United States
  • Turner, David L., University of Michigan, Ann Arbor, Michigan, United States
  • Bitzer, Markus, University of Michigan, Ann Arbor, Michigan, United States
Background

We previously determined that deletion of the miR-21 gene accentuates TGF-β induced, diabetic and aging-associated glomerular disease in mice. Because miRNA-target gene interactions are cell-type and context specific, we used independent methods to identify relevant in vivo target gene interactions for miR-21.

Methods

Candidate microRNA target gene interactions for miR-21 were identified by Pearson correlation between mRNA and microRNA abundance in micro-dissected glomeruli from patients undergoing nephrectomy at Michigan Medicine using Affymetrix ST2.1 arrays and small-RNA Illumina-truseq (n=39). Genes differentially expressed in kidney cortex of miR-21 deficient and wildtype mice were determined using Affymetrix ST2.1 arrays (n=8). Presence of microRNA target genes in the RNA-induced silencing complex (RISC) were experimentally identified using PAR-CLIP (photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation) and further validated using Bru-seq (bromouridine sequencing) in cultured human podocyte and HK2 cells. Retained candidate miR-21 target genes were further validated in kidney cortex of aged miR-21 deficient and wildtype mice (age 24-28 months; n=24) using qRT-PCR. The relevance of these genes for human pathology and phenotype was determined by association with clinical as well as glomerular and tubulointerstitial morphometric parameters as assessed through routine and immunohistochemical stains and quantitative image analysis in FFPE sections.

Results

1761 and 1309 genes exhibited significant correlation with miR-21-5p or miR-21-3p (p<0.05; FDR<0.05: 10 and 1, respectively). 1728 genes were significantly different between miR-21 WT and KO mouse kidney cortex (p<0.05). PAR-CLIP identified 752 and 687 cross-linked binding sites for miR-21-5p and miR-21-3p in 517 and 367 unique genes, respectively. Some genes, including Smad7, exhibited differential targeting by miR-21 in podocytes and HK2-cells, and after exposure to TGF-β. Of these miR-21 target genes, 15 exhibited statistically significant correlation with clinical and morphometric parameters in nephrectomies and two exhibited significant differential expression in kidney cortex of aged miR-21 mutant mice (p<0.05).

Conclusion

MicroRNA-target gene interactions are cell-type and context specific and require careful further investigations.

Funding

  • NIDDK Support