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Abstract: SA-OR024

miR-379 Knockout Mice and CLASH Technique Reveal Fis1 as a Novel miR-379 Target Related to Diabetic Nephropathy

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic

Authors

  • Kato, Mitsuo, City of Hope , Duarte, California, United States
  • Natarajan, Rama, Beckman Research Institute of City of Hope, Duarte, California, United States
Background

We recently showed that microRNAs (miRNAs) in the miR-379 megacluster and the host noncoding RNA (lncMGC) are co-regulated by endoplasmic reticulum stress, are upregulated in kidneys of diabetic mice and promote features of early diabetic nephropathy (DN). Here we hypothesized that miR-379-mutant mice created by CRISPR-Cas9 editing (miR-379-knockout, KO) are valuable tools to examine the in vivo functions of the miR-379 cluster and miR-379 in DN. Furthermore, new miR-379 targets identified using miR-379KO mouse mesangial cells (MMC) and the CLASH technique (crosslinking, ligation, and sequencing of hybrids) can uncover new therapeutic targets for DN.

Methods

Cas9 nickase and two guide RNAs (gRNAs) were injected into mouse eggs to create miR-379KO mice. To identify new miR-379 targets, UV-crosslinked RNA-protein complexes from wild type (WT) and miR-379KO mouse mesangial cells (MMC) were sonicated, immunoprecipitated (IP) with Ago2 antibody and ligated. Hybrid RNAs were subjected to RNA sequencing (CLASH).

Results

Several founders identified by PCR were crossed with WT mice, and homozygous miR-379KO mice (harboring 36bp deletion in the miR-379 locus) were obtained. Multiple miR-379 hybrid sequences were identified by CLASH and RNA-seq. Fis-1 (mitochondrial fission) was confirmed as a bonafide miR-379 target by Ago2 IP qPCR and Fis1 3’UTR reporter assays. siRNA-mediated Fis1 knockdown reduced key mitochondrial signals in MC, suggesting Fis1 is a new player in mitochondrial dysfunction in DN. Notably, miR-379-KO mice were protected from early features of DN, and Fis1 expression was decreased in kidneys of diabetic WT but not diabetic miR-379KO mice.

Conclusion

Combination of miR-379-mutant mice created by CRISPR-Cas9 editing and CLASH technique identified Fis1 as a new miR-379 target with key mitochondrial functions in the diabetic kidney. Fis1 could be a novel therapeutic target for DN.

Funding

  • NIDDK Support