MicroRNAs: Recent insights towards their role in male infertility and reproductive cancers

  • Muhammad Babar Khawar Molecular Physiology/Endocrinology Laboratory, Department of Zoology, University of the Punjab, Lahore, Pakistan State Key Laboratory of Stem Cells and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China
  • Rabia Mehmood Molecular Physiology/Endocrinology Laboratory, Department of Zoology, University of the Punjab, Lahore, Pakistan
  • Nabila Roohi Molecular Physiology/Endocrinology Laboratory, Department of Zoology, University of the Punjab, Lahore, Pakistan
Keywords: Infertility, microRNAs, miRNAs, noncoding RNAs, reproductive cancers, spermatogenesis

Abstract

Spermatogenesis is a tightly controlled, multi-step process in which mature spermatozoa are produced. Disruption of regulatory mechanisms in spermatogenesis can lead to male infertility, various diseases of male reproductive system, or even cancer. The spermatogenic impairment in infertile men can be associated with different etiologies, and the exact molecular mechanisms are yet to be determined. MicroRNAs (miRNAs) are a type of non-protein coding RNAs, about 22 nucleotides long, with an essential role in post-transcriptional regulation. miRNAs have been recognized as important regulators of various biological processes, including spermatogenesis. The aim of this review is to summarize the recent literature on the role of miRNAs in spermatogenesis, male infertility and reproductive cancers, and to evaluate their potential in diagnosis, prognosis and therapy of disease. Experimental evidence shows that aberrant expression of miRNAs affects spermatogenesis at multiple stages and in different cell types, most often resulting in infertility. In more severe cases, dysregulation of miRNAs leads to cancer. miRNAs have enormous potential to be used as diagnostic and prognostic markers as well as therapeutic targets in male infertility and reproductive system diseases. However, to exploit this potential fully, we need a better understanding of miRNA-mediated regulation of spermatogenesis, including the characterization of yet unidentified miRNAs and related regulatory mechanisms.

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MicroRNAs: Recent insights towards their role in male infertility and reproductive cancers
Published
2019-02-12
How to Cite
1.
Khawar MB, Mehmood R, Roohi N. MicroRNAs: Recent insights towards their role in male infertility and reproductive cancers. Bosn J of Basic Med Sci [Internet]. 2019Feb.12 [cited 2020Sep.30];19(1):31-2. Available from: https://www.bjbms.org/ojs/index.php/bjbms/article/view/3477
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Reviews

INTRODUCTION

About 10–15% of couples trying to conceive face infertility and male factors contribute to approximately 50% of these cases [1]. In the past several decades, considerable efforts have been made to elucidate the etiology of male infertility, however, the causative factors are still not completely understood. For example, in men with non-obstructive azoospermia (NOA) undergoing testicular sperm extraction at one center, the rates of sperm retrieval, fertilization and clinical pregnancy were lower and the frequency of chromosomal abnormalities/Y chromosome microdeletion was higher in those that had uniform maturation arrest (MA) and normal follicle-stimulating hormone (FSH), compared to other men with NOA [2]. Thus, clarifying the pathogenesis of MA may contribute to better outcomes in these patients [1].

Spermatogenesis is a multi-step process in which undifferentiated (diploid) spermatogonial stem cells (SSCs) develop into mature and motile (haploid) sperm cells called spermatozoa. The distinct stages of spermatogenesis include [3,4]: 1) The mitotic division of the SSCs which results in two types of spermatogonia; type A cells are reserved for the renewal of SSCs, while type B cells 2) further differentiate into (diploid) primary spermatocytes. The primary spermatocytes undergo meiosis I resulting in two (haploid) secondary spermatocytes and these cells, through meiosis II, produce four (haploid) spermatids. 3) In the final stage called spermiogenesis, spermatids undergo numerous morphological changes, such as the formation of the acrosome, nuclear condensation, development of the flagellum and reorganization of the cytoplasm, to give rise to mature spermatozoa (Figure 1).

FIGURE 1: A schematic representation of different stages of spermatogenesis in which mature and motile spermatozoa are produced. Spermatogonial stem cells (SSCs) develop from gonocytes in the postnatal testis, which arise from primordial germ cells (PGCs) during fetal development. In adult males, the mitotic division of the SSCs results in two types of spermatogonia; type A cells are reserved for the renewal of SSCs, while type B cells further differentiate into (diploid) primary spermatocytes. The primary spermatocytes undergo meiosis I resulting in two (haploid) secondary spermatocytes and these cells, through meiosis II, produce four (haploid) spermatids. In the final stage called spermiogenesis, spermatids undergo numerous morphological changes, such as the formation of the acrosome, nuclear condensation, development of the flagellum and reorganization of the cytoplasm, to give rise to mature spermatozoa.

Early studies indicated the role of increased apoptosis rate of spermatogonia in MA and hypospermatogenesis in infertile men, based on histological evidence and detection of apoptotic DNA fragmentation [5]. On the basis of Ki-67 and proliferating cell nuclear antigen (PCNA) immunoreactivity in testicular biopsies of oligozoospermic men with mixed atrophy, Steger at al. [6] showed that low spermatogenic efficiency in infertile men is not only associated with meiotic and postmeiotic events but also with a functional impairment of spermatogonia [6]. In addition, Maymon et al. [7] suggested that in azoospermic cases characterized by homogenous appearance of spermatocyte MA, the spermatogenic failure was a consequence of meiosis impairment (low rate of bivalent formation) rather than a malfunction of spermatogonial activity [7]. Similarly, other studies demonstrated an increased occurrence of recombination defects and meiotic abnormalities in men with NOA [8,9]. Overall, the above-described studies indicate that the spermatogenic impairment in infertile men can be associated with various etiologies, and the exact molecular mechanism underlying the regulation of spermatogonial proliferation is yet to be determined.

The aim of this review is to summarize the recent findings regarding the role of microRNAs in spermatogenesis, male infertility and reproductive cancers, and to evaluate their potential in diagnosis, prognosis and therapy of disease.

MICRORNAS: A HISTORICAL PERSPECTIVE

It is now well-established that only a small fraction of the human genome (only 1–2%) is comprised of protein-coding sequences, while the rest is made up of regulatory DNA sequences, noncoding RNA (ncRNA) sequences, introns, transposons, repeats, and of sequences whose function has yet to be determined. ncRNAs include transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), small RNAs such as microRNAs (miRNAs) and small interfering RNA (siRNAs), and long ncRNAs. miRNAs are small ncRNAs of about 22 nucleotides in length, involved in the post-transcriptional regulation through translational repression, cleavage or destabilization of the target mRNA [10-14].

Since the discovery of the first small regulatory RNAs lin-4 and let-7 in Caenorhabditis elegans, which were later classified as miRNAs, more than 1500 different miRNA genes have been identified in the human genome [15-18]. It is predicted that over 60% of human protein-coding genes are conserved targets of miRNAs [19].

In the first stages of miRNA transcription, the precursor molecules of miRNAs (primary transcripts, pri-mRNAs) are transcribed in the form of long hairpin structures, either by RNA polymerase (pol) II from independent genes (intergenic, otherwise noncoding regions) or from introns or untranslated region (UTR) of protein-coding genes. The transcription of miRNA from independent genes is regulated in a similar way as in protein-coding genes; i.e., RNA pol II, promoters, enhancers, silencing elements and various transcription factors are required to generate pri-miRNAs with a 5′ 7-methylguanosine cap and 3′ polyadenylated [poly(A)] tail. In addition, nearly half of all miRNA genes are arranged in close proximity to each other (within 50 kb of another miRNA gene) and these clusters, ranging from 2 to 46 miRNAs (the largest, chromosome 19 miRNA cluster [C19MC] in primates), can be transcribed as polycistronic units [20]. After the initial transcription, pri-miRNAs undergo two processing steps catalyzed by two enzymes from ribonuclease III (RNase III) superfamily, Drosha and Dicer. First, the nuclear Drosha-DGCR8 complex processes the newly transcribed pri-miRNA into an ~70-nucleotide intermediate termed a precursor (pre)-miRNA. This pre-miRNA is then transported to the cytoplasm where it is processed by the Dicer-TRBP complex resulting in an ~20-bp miRNA: miRNA* duplex (passenger strand), and one strand of this duplex is the mature miRNA. Following Dicer processing, the miRNA duplex is loaded onto an AGO protein to form the miRNA-induced silencing complex (miRISC). The mature miRNA strand is incorporated into the miRISC, while the other (star) strand is released from the duplex and degraded. Finally, as a part of (activated) miRISC, miRNA binds to the3′-UTR of target mRNA by recognizing mRNA sequences that are complementary to miRNA seed region (residues 2–8 at the 5′ end of miRNA) and leads to either translational repression or mRNA deadenylation and degradation of the target gene (Figure 2) [21].

FIGURE 2: MicroRNA (miRNA) biogenesis and their role in post-transcriptional regulation. In the first stages of miRNA transcription, the precursor molecules of miRNAs (primary transcripts, pri-mRNAs) are transcribed in the form of long hairpin structures, either by RNA polymerase (pol) II from independent genes (intergenic, otherwise noncoding regions) or from introns or untranslated region (UTR) of protein-coding genes. The transcription of miRNA from independent genes is regulated in a similar way as in protein-coding genes; i.e., RNA pol II, promoters, enhancers, silencing elements and various transcription factors are required to generate pri-miRNAs with a 5′7-methylguanosine cap and 3′ polyadenylated [poly(A)] tail. After the initial transcription, pri-miRNAs undergo two processing steps catalyzed by two enzymes from ribonuclease III (RNase III) superfamily, Drosha and Dicer. First, the nuclear Drosha-DGCR8 complex processes the newly transcribed pri-miRNA into an ~70-nucleotide intermediate termed a precursor (pre)-miRNA. This pre-miRNA is then transported to the cytoplasm where it is processed by the Dicer-TRBP complex resulting in an ~20-bp miRNA: miRNA* duplex (passenger strand), and one strand of this duplex is the mature miRNA. Following Dicer processing, the miRNA duplex is loaded onto an AGO protein to form the miRNA-induced silencing complex (miRISC). The mature miRNA strand is incorporated into the miRISC, while the other (star) strand is released from the duplex and degraded. Finally, as a part of (activated) miRISC, miRNA binds to the 3′-UTR of target mRNA by recognizing mRNA sequences that are complementary to miRNA seed region (residues 2–8 at the 5′end of miRNA) and leads to either translational repression or mRNA deadenylation and degradation of target gene [21].

MICRORNAS: A KEY PLAYER IN THE REGULATION OF BIOLOGICAL PHENOMENA

miRNAs contribute to the regulation of various biological processes such as embryonic development, cell differentiation, cell cycle, cell growth and apoptosis [22-24], and dysregulation of miRNA functions can lead to the development of disease. For instance, psoriasis, cardiovascular disease, schizophrenia, diabetes, cancer, kidney disease and muscular disorders have been associated with impaired biosynthesis, mutations or improper regulation of miRNAs and their targets [25-31].

Different experimental and computational approaches for identifying miRNA targets, mostly based on the patterns of complementary base pairing between miRNA and target mRNA, have demonstrated that a single miRNA can regulate the expression of hundreds of target genes [32].

miRNAs are essential for early embryogenesis and cellular differentiation, where their activity is regulated by other proteins/complexes depending on the stage. For example, miRNAs are involved in the maintenance of primordial germ cells (PGCs) through the interaction with RNA-binding protein (RBP) Dead end (Dnd1), which counteracts the suppressive function of miRNAs by preventing its association with the target mRNA [33].

Previous research demonstrated that human spermatozoa retain various RNAs (including miRNAs) after the completion of spermatogenesis; moreover, changes in the expression of spermatozoal RNAs have been associated with male infertility. Different molecular techniques such as reverse transcription polymerase chain reaction (RT-PCR), real-time PCR, complementary DNA (cDNA) cloning, microarrays, DNA and RNA sequencing are employed for the detection and analysis of these RNAs in sperm samples [34].

miRNAs are present in Leydig cells, Sertoli cells and spermatogonia within the testis, as well as in mature spermatozoa, and have a vital role in testicular development, function and spermatogenesis. The presence of miRNAs in spermatozoa confirms their role in early embryonic development after fertilization, including epigenetic effects and chromatin restructuring [35].

ROLE OF MICRORNAS IN SPERMATOGENESIS AND MALE INFERTILITY

As post-transcriptional repressors, miRNAs are important in the regulation of spermatogenesis, and the dysregulation of miRNAs has detrimental effects on male fertility [4]. Generally, the enzymes involved in the biosynthesis of miRNA- and RNA interference (RNAi)-related pathways, such as Dicer protein, appears to be crucial for normal development, as the loss of Dicer during early embryonic development in mice leads to lethality [36]. To determine the exact roles of small ncRNAs in the male reproductive system, various mouse models have been developed using the Cre/Lox system, which enables conditional manipulation of a target gene (e.g., to knock out, insert, replace, activate or modify the expression of a gene) [37-45]. Cell-specific deletion of the Dicer gene (Dicer1) in mouse epididymal cells affected differentiation of the epididymal epithelium, lipid synthesis and sperm maturation [37,38]; in Sertoli cells had a major impact on testicular proteome [40], resulted in complete absence of spermatozoa and progressive testicular degeneration [45]; in male germ cells impaired haploid spermatid differentiation [39] and led to apoptosis and/or spermatogenic failure in the meiotic and haploid phases [41,42,44]; and in PGCs affected their proliferation and spermatogenesis at early stages [43]. In most of these cases the sum of the defects led to male infertility. As demonstrated by Björkgren et al. with a conditional knock-out of Dicer1 in the proximal mouse epididymis (Dicer1fl/fl; Defb41iCre/wt [Dicer1 cKO] mice) [37,38], not only that the sperm production depends on the miRNA biogenesis, but also the sperm maturation in the principal cells of the epididymis is impaired in the absence of mature miRNAs. The sperm cells from Dicer1 cKO mice had a reduced ability to bind to and fertilize the oocyte in vitro [37].

A large number of studies have been carried out to determine the roles of individual miRNAs expressed during spermatogenesis, and their effects on male fertility [46-48]. In mice, deletion of two functionally related, miR34b/c and miR-449, clusters encoding five miRNAs impaired both meiosis and spermatozoa maturation, resulting in oligoasthenoteratozoospermia (OAT) [47]. Another study showed that simultaneous inactivation of these two miRNA clusters in double-knockout (KO) mice leads to developmental defects and infertility in males and females, which was correlated with dysregulation of more than 200 genes targeted by miR34 and miR-449 miRNAs [48]. Comparably, double KO of miR-200b and miR-429 resulted in anovulation and reduced fertility in female mice, probably by affecting the hypothalamo-pituitary-ovarian axis. On the other hand, in double KO male mice abnormalities in the testis or fertility defects were not observed [46]. Targeted deletion of specific miRNAs is a key step in understanding their function in vivo. However, a deletion of a single gene usually does not produce a recognizable phenotypic effect because of the compensatory action of related miRNAs.

Analyzing miRNA expression profiles in spermatozoa of healthy fertile men, Salas-Huetos et al. [49] showed that 221 miRNAs were consistently detected in all 10 individuals; among the 10 most expressed miRNAs hsa-miR-34b-3p was associated with the regulation of male meiosis via the E2 factor-retinoblastoma protein (E2F-pRB) pathway, hsa-miR-132-3p with cell cycle progression through MYC activation, and hsa-miR-191-5p with sperm differentiation. Moreover, hsa-miR-30b-5p, hsa-miR-200c-3p, hsa-miR-30c-5p, hsa-miR-19b-3p, hsa-miR-891a and hsa-miR-375 were related to cancer development and/or aging [49]. The first report on altered miRNA expression in patients with NOA showed that 154 miRNAs were differentially downregulated and 19 upregulated in NOA patients compared to fertile men [50]. In another study, 197 miRNAs in Sertoli cells, 68 in mixed atrophy, and 46 in spermatocyte arrest group were differentially expressed compared to samples with normal spermatogenesis. Among these, miRNAs from miR-449 and miR-34 families (e.g., hsa-miR-449a, hsa-miR-34b*, hsa-miR-34b, and hsa-miR-34c-5p), which are preferentially expressed in the testis, were validated by RT-qPCR and implicated in apoptosis, cell proliferation and differentiation according to bioinformatics analysis [51]. Overall, these results confirm the role of miRNAs in the regulation of male germ and somatic cells, and any change in their expression may lead to reproductive abnormalities [51]. Similarly, other studies reported up/downregulation of different miRNAs in asthenozoospermic, oligoasthenozoospermic, NOA, and/or teratozoospermic versus normozoospermic men, indicating their potential use in the diagnosis and treatment of male subfertility/infertility (Table 1) [52-60].

TABLE 1: Up and downregulation of various microRNAs (miRNAs) in subfertile and infertile men

In addition, specific genes known to be involved in spermatogenesis and to affect male fertility were experimentally shown to be regulated by miRNAs. Zhou et al. [61] demonstrated that the protein expression of cysteine-rich secretory protein 2 (CRISP2) is downregulated by miR-27a in asthenoteratozoospermia (ATZ) patients. Namely, they observed a negative correlation between miR-27a (high) and CRISP2 protein (low) expression in ATZ patients and showed that miR-27a binds to the CRISP2 3′-UTR mediating its translational repression. CRISP2 protein is important for sperm flagellar motility, acrosome reaction and sperm-egg fusion [61]. In oligospermic infertile patients, estrogen receptor-alpha (ERα) levels were significantly decreased compared to fertile men, while the levels of miR-100 and let-7b miRNAs were significantly higher in oligospermic vs. control group, indicating their involvement in the regulation of estrogen signaling. The important role of estrogen and estrogen receptors in the function of the male reproductive system has been indicated by experimental studies on mice, as well as genetic and clinical studies on infertile/subfertile men [62-64]. In severe oligozoospermic patients, phosphatidylinositol-specific phospholipase C, X domain containing 3 (PLCXD3), a protein which is normally expressed in the late stages of spermatogenesis, was reported to be downregulated by miR-34c-3p [65]. Another study demonstrated that, in sterile men with NOA (MA or hypospermatogenesis), miR-210 was significantly upregulated compared to control OA group, and apparently affected spermatogenesis by targeting insulin-like growth factor 2 (IGF2). Generally, IGFs control biological processes by regulating cell survival, growth, proliferation and ­differentiation, and are important for proper development/function of the testis, including the regulation of Sertoli cell number and testis size [66]. A summary of studies on the role of miRNAs in spermatogenesis is presented in Table 2.

TABLE 2: Recent findings on the role of microRNAs (miRNAs) in spermatogenesis

MICRORNAS AS A POTENTIAL BIOMARKER OF MALE INFERTILITY

In addition to cellular microenvironment, including Sertoli, Leydig cells, spermatogonia and mature spermatozoa, miRNAs can be detected in seminal and other body fluids, and these are called circulating or extracellular miRNAs. In normal physiological conditions, levels of extracellular miRNAs are stable in body fluids, and alterations in the expression of intra and extracellular miRNAs may indicate the presence of pathophysiological processes [35].

Seminal plasma is a complex fluid composed of secretions from the testis, epididymis, seminal vesicles, ampullae, prostate and bulbourethral glands. The presence of extracellular miRNAs in seminal plasma, either within extracellular vesicles (EVs) such as exosomes and microvesicles, or possibly associated with protein complexes, suggests their use as non-invasive biomarkers for spermatogenesis impairment and infertility. Both extra and intracellular miRNAs (from spermatozoa) can be isolated by non-invasive procedures and detected with routine RNA techniques such RT-qPCR and miRNA microarray, providing some advantages over conventional, invasive diagnostic procedures (e.g., prostate and testicular biopsies) [35,59,67].

Several studies reported differential expression of stable, extracellular miRNAs in seminal plasma from infertile men compared to fertile controls [57,59]. For instance, Wang et al. [59] showed that miR-34c-5p, miR-122, miR-146b-5p, miR-181a, miR-374b, miR-509–5p, and miR-513a-5p were markedly decreased in azoospermic but increased in asthenozoospermic men, compared to controls. Moreover, their ROC curve analyses indicated that the seminal plasma concentrations of these miRNAs could accurately distinguish infertile patients from fertile controls as well as azoospermic from asthenozoospermic men [59]. By analyzing miRNA profiles from normal, vasectomized and vasovasostomized donors, Belleannée et al. [68] investigated to what extent vasectomy affects miRNA expression in the epididymis and seminal microvesicles (SMVs), and whether miRNA changes in SMVs are reversible with vasovasostomy. They showed that vasectomy significantly changed the expression of epididymal miRNAs as well as affected the expression of 118 miRNAs in SMVs, including miRNAs contained in epididymosomes. Out of 118 altered miRNAs in SMVs, the expression changes were reversible for 52 and non-reversible for 66 miRNAs following vasovasostomy [68]. Most of the miRNAs responsive to vasectomy and its reversal [68] were also associated with bovine epididymosomes [69]. The exception are the members of miR-888 cluster family (i.e., miR-888, mir-890, miR-891a/b, and miR-892a/b), which are absent in all mammalian species except primates [69]. This miR-888 cluster is located on the X chromosome and expressed almost exclusively in epididymal tissues [69,70]. The rapid evolution of this cluster and tissue-specific expression suggest that it had an important role in the development of primate-specific epididymal functions. Computational functional analyses supported these assumptions as the predicted targets of miR-888 cluster family were related to epididymal morphogenesis, sperm maturation and immune cell functions; nevertheless, these findings need to be confirmed in vivo [69,71].

In mammals, sperm cells deliver to oocytes not just the paternal genome but also the paternal epigenome, which includes protein factors, DNA methylation patterns, retained histones and noncoding RNAs [72]. Yuan et al. [72] investigated the role of two miRNA clusters including five miRNAs, miR-34b/c and miR-449a/b/c, in the fertilization, first cleavage division and subsequent preimplantation in mice. These miRNAs are generally present in sperm but absent in oocytes, and all five were required for normal spermatogenesis and male fertility, i.e., miR-34b/c and miR-449 double miR-dKO mice displayed severe spermatogenic disruptions and OAT characteristics. However, it appears that these miRNAs are dispensable for fertilization and preimplantation development, as both miR-34b/c- and miR-449-null male mice were fertile; the intracytoplasmic injection of either miR-34b/c- or miR-449-null sperm and round spermatid injection of miR-dKO sperm led to normal fertilization and normal birth rate. Overall, these results indicate the functional redundancy of the two miRNA clusters in mice [72].

During mammalian spermatogenesis, a large number of mRNAs undergo post-transcriptional and translational regulation in rapidly dividing and differentiating germ cells [73], and it is now clear that miRNAs have an essential role in these regulatory mechanisms. Moreover, some miRNAs, such as hsa-miR-372 and hsa-miR-373 [74], have been identified as potential oncogenes and implicated in the development of human testicular germ cell tumors (TGCTs).

MICRORNAS IN REPRODUCTIVE CANCERS

As indicated in previous sections, miRNAs are predicted to regulate the expression of about 60% protein-coding genes in the human genome, hence their proper functioning is pivotal for normal cellular development [75]. In addition, dysregulation of miRNAs expression can lead to the development of several cancer types, including testicular cancer [75,76].

Previous studies identified specific roles of several miRNAs in spermatogenesis, e.g., miR-18a, miR-122a and miR-34 family, which were implicated in the regulation of germ cell function, cell fate determination, maintenance of undifferentiated stem cell populations, as well as cell differentiation during spermatogenesis [76,77-79]. Characterization of specific roles of miRNAs in germ cell development can be useful for the assessment of normal and disease states, such as infertility and germ cell tumors [76].

In TGCTs and other cancers, numerous studies demonstrated that miRNAs can function as oncogenes or tumor suppressors in a tumor-specific manner, suggesting their potential as diagnostic and predictive markers as well as therapeutic targets [76,80-84]. Voorhoeve et al. [74] identified miR-372 and miR-373 (miR-371-373 cluster) as oncogenes in TGCTs. These miRNAs induced proliferation and tumorigenesis of primary human cells by neutralizing tumor protein p53 signaling probably through direct inhibition of large tumor suppressor kinase 2 (LATS2) expression, a protein associated with the Ras pathway [74]. In addition, a high-throughput screen of 156 miRNAs by qPCR in a series of samples (type II and III GCTs, embryonal carcinoma-derived cell lines, and normal testis) showed that those miRNAs could be used to discriminate type II and III GCTs, as well as the histological components of type II GCTs. They also confirmed the role of the miR-371-373 cluster in tumor development [85].

Infertile men are at higher risk to develop TGCTs compared to fertile men, and miRNAs have been implicated to control the pathways connecting male infertility and tumor development [86,87]. The expression of miR-383 is downregulated in the testes of infertile men with MA. Lian et al. [87] showed that the downregulation of miR-383 and upregulation of interferon regulatory factor 1 (IRF1) in germ cells from patients with MA was associated with increased proliferative activity of cells. On the other hand, the overexpression of miR-383 in NTERA-2 testicular embryonal carcinoma cells led to the suppression of proliferation, arrest in G1-phase and induction of apoptosis. IRF1 was indicated to have a pro-mitogenic role in spermatogonia and early spermatocytes and its expression is normally downregulated by miR-383. Thus, aberrant testicular expression of miR-383 and its effects on IRF1 and IRF1 target, cell cycle-related, genes suggest a possible connection between male infertility and TGCT [87].

It is also noteworthy to mention significant findings regarding miRNA role in prostate cancer (PCa), especially the diagnostic and prognostic value of circulatory miRNAs in PCa patients [88]. Numerous studies identified individual miRNAs that are up or downregulated in PCa patients compared to healthy controls, and notably, miR-141, miR-200b and miR-375 were identified to correlate with disease severity and could potentially be used to identify PCa patients with previously undetectable micrometastases [88]. A complex interaction between miRNA expression, androgen signaling and key pathways in prostate tumorigenesis was suggested [89]. While some miRNAs, such as miR-125b [90], miR-21, miR-32, miR-27a and miR-141, were shown to be regulated by androgen-receptor (AR) mediated signaling, others (e.g., miR-221, miR-222, miR-331-3p and miR-let7c) were suggested to modulate the AR signaling pathway function [89].

Todorova et al. [91] investigated the switch of miRNAs function (and related transcription factors) from initial tumor-suppressor to pro-oncogenic activity, due to genomic instability and rearrangement events in cancer cells. They used several PCa cell lines as models for lymph-node and bone marrow metastases and analyzed the relationship between miR-204, known for its dualistic role in cancer, and the transcriptional factors c-MYB (v-myb avian myeloblastosis viral oncogene homolog), RUNX2 (runt-related transcription factor 2) and ETS1 (v-ets erythroblastosis virus E26 oncogene homolog 1) involved in malignant lineage development, epithelial–mesenchymal transition (EMT) and metastasis. They showed dualistic effects of miR-204 on the expression of transcription factors in PCa cells, acting either as a tumor-suppressor (on c-MYB) or as an oncogene (on ETS1), which was related to DNA methylation patterns and the presence of TMPRSS2-ERG gene fusion in PCa cells (a fusion between androgen-sensitive promoter of the transmembrane protease serine 2 gene [TMPRSS2] and the v-ets erythroblastosis virus E26 oncogene homolog gene [ERG] belonging to ETS family transcription factors) [91]. Furthermore, these authors showed that miR-204 upregulates AR and downregulates TMPRSS2/ERG by directly modulating the methylation of their promoters and through related transcription factors, which prevents TMPRSS2/ERG overexpression in PCa [92]. Hsu et al. [93] described the oncogenic function of another miRNA, miR-18a, in PCa. miR-18a was significantly upregulated in their clinical PCa specimens and cancer cell lines, compared to normal controls. Their experiments with PCa cell lines and mouse model indicated that miR-18a promotes the tumorigenicity of PCa cells, by suppressing the serine/threonine-protein kinase 4 (STK4)-mediated AKT apoptosis pathway [93]. Overall, these studies indicate that modulating disrupted miRNA/target protein interactions in cancer patients may be a promising novel therapeutic strategy.

Depending on miRNA function/effects in cells, two main modes of their targeted modulation in cancer and other diseases have been proposed; i.e., either to restore/induce tumor-suppressive miRNAs or to inhibit those with oncogenic function [94]. Nevertheless, several issues must be overcome before exploring the full potential of miRNAs in the treatment of disease. These, among others, include the delivery and high expense of artificially modified nucleic acids. For example, frequent injections of miRNAs may not be a suitable option for the treatment of sub/infertility in otherwise healthy individuals, although it could be used in the treatment of disease. Also, delivering specific miRNAs to targeted tissues remains a challenge, most importantly because these miRNAs may have different functions in different tissues [76].

CONCLUSION AND FUTURE PERSPECTIVE

Understanding regulatory mechanisms involved in spermatogenesis is of utmost importance for developing effective therapeutic strategies to treat male infertility and diseases of male reproductive system. Over the past decade, a growing number of studies have demonstrated that non-protein coding RNAs, such as miRNAs, play an essential role in the regulation of biological processes, including spermatogenesis. Experimental evidence shows that aberrant expression of these miRNAs affects spermatogenesis at multiple stages and in different cell types, most often resulting in infertility. In more severe cases, dysregulation of miRNAs leads to cancer. Thus, miRNAs have enormous potential to be used as diagnostic and prognostic markers as well as therapeutic targets in male infertility and male reproductive system diseases. To exploit this potential fully, we need a better understanding of miRNA-mediated regulation of spermatogenesis, including the characterization of yet unidentified miRNAs and related regulatory mechanisms. Continuous technological advances, especially in omics technologies, may provide the necessary means to accomplish these challenging tasks.

Acknowledgements

ACKNOWLEDGMENTS

The authors are thankful to the Vice Chancellor, University of the Punjab, Lahore, Pakistan for providing financial support for the accomplishment of this review.

DECLARATION OF INTERESTS

The authors declare no conflict of interests.

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