Screening for miRNAs and their potential targets in response to TGF-β1 based on miRNA microarray and comparative proteomics analyses in a mouse GC-1 spg germ cell line.

Abstract

Transforming growth factor-β1 (TGF-β1) is a member of the TGF-β superfamily that performs a number of cellular functions and shows differential activity at different testicular developmental stages. In the present study, we investigated the effects of exogenous TGF-β1 on global microRNA (miRNA or miR) expression profiles by miRNA microarray analysis and the alterations in protein profiles by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) in a mouse GC-1 spg germ cell line. A total of 24 differentially expressed miRNAs, including 7 upregulated and 17 downregulated miRNAs were identified. The results obtained by the RT-qPCR analysis of 10 selected differentially expressed miRNAs were in accordance with those obtained by miRNA microarray analysis. In addition, 11 differentially expressed proteins, including 3 upregulated and 8 downregulated proteins were identified through MS-based comparative proteomics analysis. Bioinformatics analysis predicted that peptidyl‑prolyl isomerase A (PPIA) and nucleoside diphosphate kinase B (NDKB) are targets of miR-149 and miR-199a-3p, respectively in response to the stimulation of mouse GC-1 spg germ cells with TGF-β1. RT-qPCR revealed that the expression levels of these miRNAs showed an opposite trend in response to stimulation with TGF-β1. In conclusion, we identified some important miRNAs and proteins as possible targets involved in TGF-β1 signaling. Our data suggest the existence of a TGF-β1‑miR‑149-PPIA or TGF-β1-miR-199a-3p-NDKB pathway in GC-1 spg cells. Further studies are warranted to ascertain the role of these miRNAs in spermatogenesis.