Introduction
MicroRNAs (miRNAs/miRs) are non-coding RNAs which consist of ~20 nucleotides and have been determined to be critical regulators of eukaryotic gene expression such as in mammals, plants and viruses (1,2). The first miRNA was found >30 years ago in the nematode Caenorhabditis elegans, with the detection of the developmental regulator, lin-4 (3). To date, >2,000 miRNAs have been identified in humans governing one third of the genes in the genome (4). During synthesis, RNA polymerase II transcribes a miRNA gene to produce a long primary miRNA (pri-miRNA), which is cleaved to generate precursor miRNA (pre-miRNA) within the nucleus (5,6). The pre-miRNA is then translocated to the cytoplasm (7-9) where it is cleaved to generate a ~22 nt miRNA duplex (10,11). Of note, one strand of this duplex generates the mature guide miRNA, which binds to an Argonaute protein to become the core of the miRNA-induced silencing complex (miRISC) (12,13). The miRNA region then drives miRISC to complementary sites to govern repression of the target mRNA, while another strand of the mature miRNA duplex, as passenger (opposite) strand, degrades. In mammals, the miRNA target sites, are usually localized in the 3'-untranslated region (3´UTR) of mRNAs, which are known as the sites with the most complementarity to the seed region (14,15). The determination of miRNA-mRNA target interactions is critical for exploring the regulatory role of miRNAs, resulting in the discovery of novel therapies (16).
In recent years, the regulatory roles of miRNAs in various pathologies, ranging from cancer to autoimmune and cardiovascular disease, have attracted major attention (17). By these means, miRNAs possess promising potential for use as biomarkers in diagnostic applications. For example, following the discovery of the regulatory role of miRNAs in cancer, a number of investigations have focused on exploring their potential therapeutic value (18). Currently, the Food and Drug Administration and the European Medicines Agency have accepted a total of 11 RNA-based therapeutics involving small interfering RNA and antisense oligonucleotide. Although miRNAs are not yet approved as therapeutic agents for diseases, some miRNA mimics and anti-miR oligonucleotides are undergoing or completed clinical phase I (NCT01829971, NCT04675996, NCT02369198, NCT04045405) and II (NCT03837457, NCT03373786, NCT02855268 and NCT03601052) trials. For instance, an anti-miR-122 drug, miravirsen, developed for treatment of hepatitis C, has successfully completed clinical phase I (NCT01646489) and II (NCT02508090, NCT02508090, NCT02452814 and NCT02452814) trials (18).
The present systematic review explored what is known regarding miRNAs and hypospadias. Hypospadias is a malformation of the male external genitalia (18.6-80 per 10,000 male births), and it is characterized by a proximal dislocation of the urethral meatus, ventrally split foreskin, and can be combined with penile curvature and transposition of the scrotum (19,20). The moderate to severe cases account for 30%, while the remaining 70% are considered a mild form that is not linked with other urogenital malformations (20).
The etiology of hypospadias is described as complex, meaning that it occurs due to genetic factors in combination with environmental factors. The most well-established risk factor is a low birth weight and growth retardation during gestation (21). In ~10% of cases, hypospadias can be found in an additional family member. Some level of genetic predisposition is established in hypospadias (7% of cases having affected first, second, or third-degree relatives) and hereditary occurrence appears more frequent for distal and middle forms than for posterior varieties (22). The risk of a male sibling also having hypospadias is 9-17% and hypospadias is evenly transmitted through the maternal and paternal side, with an assessed heritability of 57-77% (23). In only 1/3 of hypospadias, an obvious genetic reason is established; however, hypospadias has been described in >200 syndromes, with the most well-known being Wilms' tumor, aniridia, genitourinary malformations, mental retardation (WAGR) and genitourinary malformations and susceptibility to Wilms' tumor (Denys-Drash syndrome) (22,24).
In the present study, it was hypothesized that the genetic predisposition to hypospadias may not be primarily related to mutations in gene-coding regions, but may be caused by silencing intracellular post-transcriptional factors, such as miRNAs. Thus, a systematic review was performed to further elucidate what is known on the subject.
Discussion
The main purpose of the present systematic review was to explore what was already described regarding the role of miRNAs in hypospadias. The literature was analyzed systematically and a total of nine different miRNAs were determined in the nine selected articles.
In one of the studies, Tian et al (31) indicated that miR-494 decreased the expression of Neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4L), resulting in the activation of the TGF-β1/Smad signaling pathway and the promotion of hypospadias. Accordingly, Liu et al (38) demonstrated that the higher expression of TGF-β1 promoted the occurrence of hypospadias in DEHP-treated mice. Of note, other studies have demonstrated the essential role of the TGF-β1 signaling pathway in urethral development (38-40). Moreover, the TGF-β signaling pathway and its downstream genes, including activating transcription factor 3, connective tissue growth factor and cysteine-rich angiogenic inducer 61, have been suggested as potential factors in the etiology of hypospadias (38). TGF-β1 governs a vast range of biological processes, such as proliferation, EMT and apoptosis (41-43). EMT is a critical event in urethral embryogenesis (44,45). During this critical biological process, epithelial cells achieve mesenchymal features, such as becoming non-polarized, losing intercellular adhesions and moving throughout the extracellular matrix for tissue generation (46). Previous studies have suggested the transformation of urethral epithelial cells into mesenchymal cells following urethral plate fusion through EMT (44,46). The disruption of the EMT process can cause a failure of the urethral plate fusion, leading to the occurrence of hypospadias (46-48). A recent study determined that miR-1199-5p targets SRD5A2, influencing EMT transformation and promoting the development of hypospadias, highlighting the crucial role of EMT through miR-1199-5p in this condition (33). Moreover, TGF-β1 signaling also plays a critical role in hypospadias development by its regulation of EMT. Notably, recent studies have demonstrated miR-494 as a key regulator of TGF-β1 signaling (49-51).
Previous studies have also demonstrated that miRNAs play a critical role in the regulation of Nedd4L expression, which mediates TGF-β1 signaling (52,53). It has been shown that Nedd4L is a critical ubiquitin ligase which selectively targets the activation of Smad2/3 for degradation, resulting in the suppression of TGF-β1 signaling (52). Therefore, miR-494 can indirectly regulate the occurrence of hypospadias. Accordingly, Qian et al (32) confirmed the importance of the TGF-β1 signaling pathway in the occurrence of hypospadias through the regulatory role of miR-200c by comparing a rat model of hypospadias induced by DEHP administration with healthy rats. They indicated that there were low levels of miR-200c expression in hypospadias penile tissues, resulting in the high expression of the Zeb1 gene and protein, thereby activating the TGF-b/Smad3 pathway (32). However, both studies included a small sample size, and the degree of hypospadias was not specified in their findings.
miR-145 has been suggested as another potential regulator for the progression of hypospadias in the study by Shang et al (30), one of the studies selected for the present systematic review. Previous studies have demonstrated the regulatory role of miR-145 in the expression of pluripotency factors, leading to the reduction of self-renewal and promoting the differentiation of human embryonic cells (54,55). In addition, miR-145 has been demonstrated to affect embryo attachment by reducing the expression of maternal IGF1R. miR-145 has also been shown as a suppressor of phosphorylated extracellular-regulated kinase expression by targeting PAK4, resulting in the reduction of human colorectal cell growth (56). The importance of mitogen-activated protein kinase (MAPK) signaling expands to spermatogenesis and dysgenesis activated by environmental toxicities. This can be ascribed to the compromised blood-testis barrier, leading to reduce semen quality, which could also serve as a contributing factor to hypospadias (57). Of note, MAPK signaling can alter the proper action of downstream SRY-Box transcription actor 9 (SOX9) and the mutation of SOX9 can progress the development of hypospadias (58). SOX9 was found to increase testis development in transgenic mice with XX karyotype (59). Furthermore, the mutation of SOX9 can enhance the progression of campomelic syndrome and sex reversal of XY (60). All the effects of SOX9 on testis development, sex differentiation and hormone secretion suggest a linkage between SOX9 and hypospadias development. Of note, SOX9 has been demonstrated as a target gene of miR-145 for regulating cell activities. Moreover, several studies have shown the accelerating effects of miR-145 on cell apoptosis (61-64). These studies have revealed its inhibitory roles on the proliferation, migration and invasion of the cells through multiple targets, such as Mucin 1, c-Myc, Kirsten rat sarcoma virus, Bax and caspase-3 (61,62).
Cell apoptosis has been demonstrated as a key criterion for normal embryogenesis of male genital tubercles and the anterior urethra. Furthermore, the abnormal alteration of apoptosis in the urethra of mice has been reported in mouse models of hypospadias. Therefore, miR-145 may progress the development of hypospadias through aberrant cell apoptosis. Surprisingly, Shang et al (30) suggested the regulatory role of miR-145 in the development of hypospadias was through MAPK signaling and SOX9 expression. They also demonstrated the inhibitory role of miR-145 on nuclear factor erythroid 2-related factor 2-HO-1 signaling and downstream glutathione peroxidase 1 and superoxide dismutase type 1 expression, demonstrating the role of miR-145 as a contributor to hypospadias through the suppression of the antioxidant system. A recent study confirmed the theory of hypospadias development through apoptosis by demonstrating the regulatory role of miR-556 in the GATA4 expression level (29). GATA4 has also been shown as a regulator of cell survival (anti-apoptotic) signaling (65). This is supported by findings from the study by Grepin et al (66), where the depletion of GATA4 led to apoptosis. These studies demonstrate the involvement of both miR-145 and miR-556 in the development of hypospadias through apoptosis.
miR-6756-5p has been proposed as a potential biomarker involved in the progression of hypospadias. Huang et al (25) demonstrated that a high level of miR-6756-5p led to AR suppression, enhanced the activation of PI3K/AKT pathway, the proliferation of HFF-1 cells and impaired apoptosis, potentially contributing to the development of hypospadias. However, the precise functional impact of these molecular events and cellular responses on the progression of hypospadias was not examined in their study. This is a significant limitation as without this information, a direct link between the two cannot be definitively established. Of note, previous studies have indicated that patients with severe hypospadias exhibit a defect in the AR gene (67-70). Specifically, there is evidence suggesting that a crucially reduced level of AR mRNA expression could play a significant role in the development of hypospadias at the midshaft (67). Therefore, miR-6756-5p may play a pivotal role as a post-transcriptional regulator of AR in genital mesodermal cells, which could potentially contribute to hypospadias driven by AR loss in humans. Peng et al (28) treated HFF-1 cells with testosterone and revealed that miR-143-3p, by targeting IGFBP-3, impede cell growth and decrease AR signaling, thereby counteracting testosterone, leading to the progression of hypospadias. Therefore, miR-143-3p can be another potential regulator of AR signaling, resulting in the regulation of hypospadias development. Notably, it should be considered that their study did not explore the detailed mechanism or the direct connection between AR signaling and the progression of hypospadias. Furthermore, all their findings were at the cellular level and require validation in human and animal samples. miR-143-3p has also been shown to modulate specific target genes associated with cancer, cardiovascular disease and other health conditions, influencing the progression of these diseases in numerous studies (71-74). Furthermore, studies have indicated that IGFBP-3 plays a role in influencing the progression of various diseases (75-77). This is achieved by inhibiting receptor binding and impeding their anti-apoptotic functions. Additionally, IGFBP-3 has been reported to hinder angiogenesis and impact apoptosis (78-80).
Deng et al (26) also discovered that miR-182 can target the seeding region encompassing rs3743104 within the 3'-UTR of GREM1, which in turn inhibits GREM1 transcription. miR-182 has previously been documented as a biomarker for kidney injury and bladder cancer (81,82). It functions as a regulatory miRNA by binding to the 3'-UTR of target genes, influencing the progression of diseases (81). GREM1 is recognized as part of the bone morphogenic protein antagonist family (BMP) (82), and has been linked to susceptibility to hypospadias. Although these findings did not demonstrate a direct link between miR-182 and the progression of hypospadias, miR-182 could potentially play a key role in advancing the development of hypospadias through the BMP signaling pathway.
miR-210 may serve as another regulatory key in the onset of hypospadias. Elias et al (27) measured the plasma level of miR-210 in patients with 46,XY DSD with atypical genitalia, demonstrating a higher expression of miR-210 in hypospadias compared with the control group. It has been shown that miR-210 directly targets IGF2 through in vitro experiments conducted in NT2 cells. Consequently, these findings suggest a potential association between miR-210 and Insulin/IGF signaling. However, it should be considered that their study involved a small group of patients. Prior research has demonstrated that miR-210 expression is elevated in the testes of infertile men with maturation arrest (83,84). Moreover, miR-210 exhibits a ubiquitous expression across a diverse range of cells, playing physiological roles, such as suppressing cell proliferation, mitochondrial respiration and DNA repair, and affecting vascular biology and angiogenesis (85-87). Recognized as a major hypoxia-inducible miRNA, its relevance is noteworthy in conditions, such as placental hypertension and preeclampsia, which can induce fetal hypoxia (88). A previous study demonstrated that insulin/IGF and hypoxia signaling act in concert in Caenorhabditis elegans (89). Therefore, miR-210 can promote the development of hypospadias through insulin/IGF1 and hypoxia signaling.
Surprisingly, several miRNAs in the reviewed articles, including miR-494, miR-145, miR-6756-5p, miR-182, miR-210 and miR-200c, were found to be associated with key clinical features of hypospadias, such as disease severity and specific anatomical abnormalities. miR-494 was found to be associated with more severe forms of the condition, potentially due to its impact on TGF-β1/Smad signaling pathways. Moreover, the downregulation of miR-200c was linked to impaired EMT processes in urethral development. To investigate the role of miRNAs in the clinical presentation of hypospadias, the association between specific miRNAs and key clinical characteristics was analyzed. The analysis suggested that specific miRNAs could serve as biomarkers for predicting the severity and anatomical characteristics of hypospadias. Further studies with larger sample sizes are required however, to validate these findings and explore the direct mechanistic roles of these miRNAs in hypospadias.
The present systematic review provides a comprehensive analysis of the role of specific miRNAs in the development of hypospadias, integrating evidence from both human and animal studies. By highlighting miRNAs, such as miR-494, miR-145, miR-6756-5p, miR-182, miR-210, miR-143-3p, miR-556 and miR-200c, the present systematic review identifies key molecular pathways, including TGF-β, MAPK, PI3K/AKT, BMP and AR signaling (Fig. 2), that are crucial in regulating cellular processes, such as proliferation, apoptosis and differentiation resulting in hypospadias progression. The findings suggest that miRNAs do not merely function as biomarkers, but actively participate in disease progression, linking genetic predispositions with environmental influences. This integrative approach provides valuable insight into the molecular mechanisms underlying hypospadias and underscores the potential of miRNAs as therapeutic targets, setting the stage for future translational research aimed at developing novel diagnostic and treatment strategies.
However, the included studies faced significant limitations, including small sample sizes, a lack of stratified analyses, the absence of specific treatment groups and findings limited to cellular level, which restricted the understanding of the molecular mechanisms and the direct links between miRNAs and hypospadias progression. Further investigations are required to validate these findings in human and animal samples and to explore the detailed roles of miRNAs in the development of hypospadias. A limitation of the present systematic review is that the literature search was conducted using only two databases, which may have restricted the comprehensiveness of the review and may have thus potentially excluded relevant studies. Several potential biases in the included studies have been highlighted throughout the present systematic review. Small sample sizes reduce the statistical power and limit generalizability, while selection bias arises from focusing on specific populations, such as pediatric patients and animal models. Language bias from including only studies in the English language and publication bias favoring significant results may skew the findings. Variability in experimental methods and the lack of stratified analyses further complicate comparisons across studies. The majority of the studies were observational, lacked validation in human samples, and often relied on single time point measurements, which may miss dynamic changes. Additionally, confounding factors, such as environmental exposures and genetic variability were not consistently controlled, limiting the ability to establish the link between miRNAs and hypospadias.
In conclusion, the present systematic review demonstrated the link between miR-494, miR-145, miR-6756-5p, miR-182, miR-200c, miR-210 and miR-1199-5p, and the occurrence of hypospadias. The majority of the included articles focused on etiological factors. Notably, miR-494, miR-200c, miR-145, miR-6756-5p, miR-182 and miR-210 could promote the development of hypospadias through different signaling pathways, such as TGF-β, MAPK, PI3K/AKT, BMP, insulin/IGF and hypoxia. These pathways are also involved in tissue regeneration and wound healing. Therefore, the present systematic review suggests that miRNAs may serve as potential biomarkers related to hypospadias and may play a role in potentiating wound healing following surgery. Further investigations are required however, to shed light onto the regulatory role of miRNAs in the development of hypospadias, which can provide a novel direction for the understanding of the etiology of hypospadias and improving treatment modalities.