Abstract
Our previous studies have shown that microRNA-383 (miR-383) expression is downregulated in the testes of infertile men with maturation arrest (MA). However, the underlying mechanisms of miR-383 involved in the pathogenesis of MA remain unknown. In this study, we showed that downregulation of miR-383 was associated with hyperactive proliferation of germ cells in patients with mixed patterns of MA. Overexpression of miR-383 in NT2 (testicular embryonal carcinoma) cells resulted in suppression of proliferation, G1-phase arrest and induction of apoptosis, whereas silencing of miR-383 reversed these effects. The effects of miR-383 were mediated through targeting a tumor suppressor, interferon regulatory factor-1 (IRF1), and miR-383 was negatively correlated with IRF1 protein expression in vivo. miR-383 inhibited IRF1 by affecting its mRNA stability, which subsequently reduced the levels of the targets of IRF1, namely cyclin D1, CDK2 and p21. Downregulation of IRF1 or cyclin D1, but not that of CDK2, enhanced miR-383-mediated effects, whereas silencing of p21 partially inhibited the effects of miR-383. Moreover, miR-383 downregulated CDK4 by increasing proteasome-dependent degradation of CDK4, which in turn resulted in an inhibition of phosphorylated retinoblastoma protein (pRb) phosphorylation. These results suggest that miR-383 functions as a negative regulator of proliferation by targeting IRF1, in part, through inactivation of the pRb pathway. Abnormal testicular miR-383 expression may potentiate the connections between male infertility and testicular germ cell tumor.
Highlights
(MA, a cause of nonobstructive azoospermia (NOA)), usually have a significantly lower sperm retrieval rate and a clinical pregnancy rate.[2]
Real-time PCR results suggested the upregulation of proliferating cell nuclear antigen (PCNA) mRNA in some patients with maturation arrest (MA) (Figure 1c), specimens obtained from men with the Sertoli-cell-only (SCO) syndrome showed no PCNAimmunopositive staining (Figure 1b)
Our results revealed that mRNA (Figure 6a) and protein (Figure 6b) levels of three cell cycle-related genes, namely Cyclin D1,18 CDK219 and p21,20 were all lower in NT2 cells transfected with si-interferon regulatory factor-1 (IRF1) than in cells transfected with negative controls (NCs)
Summary
(MA, a cause of NOA), usually have a significantly lower sperm retrieval rate and a clinical pregnancy rate.[2]. Some histological studies have suggested the existence of accelerated germ cell apoptosis[3] and decreased mitotic activity of spermatogonia[4] in infertile men with MA at the spermatocyte stage. The loss of Dicer[1] (an RNase III enzyme required for miRNA processing) in primordial germ cells and spermatogonia resulted in defective proliferation;[10] in Dicer-deleted testis, spermatogenesis was arrested at an early stage during proliferation and/or early differentiation,[10,11] miR-372 and miR-373 have been. We have identified many deregulated miRNAs in testicular tissues of NOA patients with MA.[13] miR-383, the expression of which is restricted to the spermatogonia and early spermatocytes, exhibited a significant downregulation of gene expression in MA patients.[13] These findings suggest that miR-383 may act on early stages of spermatogenesis and may regulate germ cell proliferation or death. MiR-383 was functionally characterized in the testicular embryonal carcinoma cell line NTERA-2 (NT2) and during spermatogenesis by identifying its target genes
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