Presentation Authors: Ryan Flannigan*, Vancouver, Canada, Russell Hayden, Anna Mielnik, Alex Bolyakov, Peter Schlegel, Darius Paduch, New York, NY
Introduction: Our group has previously demonstrated that men with Sertoli Cell Only Syndrome (SCO) demonstrate a 17x downregulation of miR-202-5p compared to testis biopsies from normal controls. We subsequently demonstrated that using an inducible model of SCO in mice does not deplete miR-202-5p levels. In this study, we sought to computationally evaluate mRNA targets for miR-202-5p, test for miR-202-5p to 3UTR interactions, and evaluate the impact of in vivo delivery of a miR-202-5p inhibitor on spermatogenesis.
Methods: Computationally predicted mRNA targets of miR-202-5p were created using RNAseq and miRNAseq biological data from 44 testis biopsies. A custom multi-parametric, multi-dimensional association of vectors was used to capture both linear and non-linear algorithms via neural network machine learning. To validate miR-202-5p interactions with the 3UTR of CHEK1 gene, a luciferase reporter assay was used. These reporter plasmids were transfected and later transduced in Human Embryonal Kidney (HEK293) cells prior to measuring luciferase activity in the presence of miR-202-5p. To test the in-vivo impact, 50nM FITC-labelled miR-202-5p inhibitor was injected into murine efferent ducts along with a scrambled control miRNA on the contralateral side. Testes were subsequently evaluated via histology and immunofluorescence.
Results: MiR-202-5p was found to be heavily implicated in epidermal growth factor (EGF) pathway regulation and cell cycle control. The computationally derived results were validated in vitro with using miRNA-3UTR luciferase reporters where miR-202-5p reduced the luciferase activity of cells transduced with CHEK1 3UTRs but not when the 3UTR was designed with a mutated seed region. We further confirmed the critical role miR-202-5p exerts during spermatogenesis by observing a knockdown of spermatogenesis in murine testis tubules in the presence of a miR-202-5p inhibitor, and maintenance of spermatogenesis in the presence of a scrambled control miRNA.
Conclusions: In follow-up to our previous studies investigating the role of miR-202-5p, we demonstrated that miR-202-5p is largely implicated in EGF pathway regulation in Sertoli cells maintaining an end-differentiated state. Furthermore, we demonstrated that miR-202-5p is critical to spermatogenesis as in vivo inhibition results in spermatogenic knockdown and SCO histopathology. Further research is prudent to determine the role of miR-202-5p therapy to potentially restore spermatogenesis in men with non-obstructive azoospermia.
Source of Funding: This work was supported by: P50 HD076210, U1 1U01HD074542-01, Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust, the Mr. Robert S. Dow Foundation, Irena and Howard Laks Foundation; Urology Care Foundation Research Scholar Award Pro