Presentation Authors: Russell Hayden*, Anna Mielnik, Peter Schlegel, Darius Paduch, New York, NY
Introduction: Chromatin-modifying complexes (CPCs) play a critical role in epigenetic gene regulation and are thought to implement global genetic programs. Several long non-coding RNAs (lncRNAs) have been found to interact with CPCs to help guide these complexes to appropriate gene targets, the most notable examples being Xist and HOTAIR. In this study, we attempted to identify CPC associated lncRNAs that were differentially expressed among men with Sertoli Cell Only Syndrome (SCO) and men with normal spermatogenesis.
Methods: Testis biopsies were obtained during testicular sperm extraction for infertility. RNA-seq was performed on testis biopsies from 11 men with SCO and 10 with normal spermatogenesis using the Illumina HiSeq2000 platform. Reads were mapped using the STAR Aligner v2.5 against human genome hg38. Raw counts were normalized with Limma v3.6. Differentially expressed lncRNAs were then screened for documented CPC interactions. Differential expression of candidate lncRNAs were then confirmed with real time PCR (RT-PCR). RT-PCR was conducted on 3 men with SCO and 3 normal controls on a LightCycler 480 (Roche). The RT2 qPCR platform was used in all experiments (Qiagen), and all assays were conducted in triplicate. Quality control was assured using melting point analysis, interplate calibrators, and negative controls.
Results: Three lncRNAs demonstrated significant differential expression, documented CPC interaction, and meaningful expression levels in normal controls. RNA-seq demonstrated a Log2 fold change of -3.8 (p < 0.001), -3.5 (p < 0.001), and -9.4 (p < 0.001) for transcripts linc00467, linc00958, and linc01016, respectively. RT-PCR confirmed downregulation, with Log2 fold changes of -2.8 (CI -3.5 to -2.1), -7.2 (CI -8.5 to -6.0), and -12.4 (CI -14.1 to -10.6).
Conclusions: Men with SCO exhibit significant down regulation of linc00467, linc00958, and linc01016. These lncRNAs have been shown to interact with CPCs and may be key mediators in master gene programs. Further study is needed to delineate how these three transcripts participate in the pathophysiology of SCO.
Source of Funding: 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.