In Silico Analysis of miRNA-Mediated Genes in the Regulation of Dog Testes Development from Immature to Adult Form

Simple Summary The objective of this investigation was to elucidate the association of miRNA-mediated genes in the regulation of dog testes development from immature to adult form by in-silico analysis. In silico analysis of differentially expressed (DE) testis miRNAs between healthy immature and mature dogs were performed using miRNet, STRING, and ClueGo programs. The determination of mRNA and protein expressions of predicted pivotal genes and their association with miRNA were studied. The predicted genes are involved in the governing of several key biological functions (cell cycle, cell proliferation, growth, maturation, survival, and apoptosis) in the testis as they evolve from immature to adult forms, mediated by several key signaling pathways (ErbB, p53, PI3K-Akt, VEGF, and JAK-STAT), cytokines and hormones (estrogen, GnRH, relaxin, thyroid hormone, and prolactin). Elucidation of DE-miRNA predicted genes’ specific roles, signal transduction pathways, and mechanisms, by mimics and inhibitors, which could perhaps offer diagnostic and therapeutic targets for infertility, cancer, and birth control. Abstract High-throughput in-silico techniques help us understand the role of individual proteins, protein–protein interaction, and their biological functions by corroborating experimental data as epitomized biological networks. The objective of this investigation was to elucidate the association of miRNA-mediated genes in the regulation of dog testes development from immature to adult form by in-silico analysis. Differentially expressed (DE) canine testis miRNAs between healthy immature (2.2 ± 0.13 months; n = 4) and mature (11 ± 1.0 months; n = 4) dogs were utilized in this investigation. In silico analysis was performed using miRNet, STRING, and ClueGo programs. The determination of mRNA and protein expressions of predicted pivotal genes and their association with miRNA were studied. The results showed protein–protein interaction for the upregulated miRNAs, which revealed 978 enriched biological processes GO terms and 127 KEGG enrichment pathways, and for the down-regulated miRNAs revealed 405 significantly enriched biological processes GO terms and 72 significant KEGG enrichment pathways (False Recovery Rate, p < 0.05). The in-silico analysis of DE-miRNA’s associated genes revealed their involvement in the governing of several key biological functions (cell cycle, cell proliferation, growth, maturation, survival, and apoptosis) in the testis as they evolve from immature to adult forms, mediated by several key signaling pathways (ErbB, p53, PI3K-Akt, VEGF and JAK-STAT), cytokines and hormones (estrogen, GnRH, relaxin, thyroid hormone, and prolactin). Elucidation of DE-miRNA predicted genes’ specific roles, signal transduction pathways, and mechanisms, by mimics and inhibitors, which could perhaps offer diagnostic and therapeutic targets for infertility, cancer, and birth control.


Introduction
MicroRNAs (miRNAs) play a key role in the differentiation, development, maintenance, and functions of various tissues. Spermatogenesis is a sequence of complex processes [1,2], which are regulated by pathways mediated by miRNAs [3,4]. The genome of testicular cells is actively transcribed into RNAs that involves many non-coding RNAs consisting of circular RNAs (circRNAs) and miRNAs to regulate and generate phasespecific gene expression patterns [5,6]. In mouse testis, pachytene, round, and elongated spermatocytes showed the highest levels of miRNA expressions [7][8][9]. Numerous miRNAs are preferentially expressed in the testis and male germ cells of humans and mice [5][6][7][8][9]. However, the biological functions of many miRNAs involved in spermatogenesis and testicular function are largely unknown. It should be noted that diminution of the Dicer gene upsets the proliferation and differentiation of mouse spermatogenic germ cells [10][11][12].
Dicer is an endonuclease enzyme that belongs to the ribonuclease III (RNase III) family. It activates the RNA-induced silencing complex (RISC), which is essential for RNA interference. The RISC contains dsRNA binding proteins, including protein kinase RNA activator (PACT) and transactivation response RNA binding protein (TRBP) that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. Dicer plays an important role in spermatogenesis [13,14]. MiRNAs along with dicer act as a post-transcriptional regulatory unit in testicular tissue development and spermatogenesis [15,16]. Therefore, miRNAs can be targeted for evaluating male fertility and can serve as useful biomarkers.
It is conceivable that miRNAs can regulate meiosis and thus spermatogenesis [17] by regulating mRNA degradation and disrupting mRNA translation [18,19]. While recent studies focused on miRNA tissue expression in rodents and humans, miRNA data for dogs are lacking [20,21]. We recently reported miRNA expression patterns between sexually immature and mature canine testes [20].
The objective of this investigation was to elucidate miRNA-mRNA interaction of differentially expressed (DE) miRNAs between immature and mature canine testis by constructing a protein-protein network and performing cluster gene analysis to elucidate key biological functions.

Conserved Nucleotide Sequences
Nucleotide sequences of differentially expressed canine miRNAs were retrieved from miRBase, (www.mirbase.org) (accessed on 4 March 2022) and compared with those of homo sapiens for similarities [22,23].

Identification of Target Genes of Differentially Expressed miRNAs
The target and predicted genes of DE-miRNAs were retrieved using miRNet (http: //www.mirnet.ca/) (accessed on 4 March 2022) [24,25]. This tool integrated data from different miR databases (TarBase, miRTarBase, and miRecords) and identified target and predicted genes. The integration analysis was performed separately for upregulated and downregulated DE-miRNAs.

Gene Ontology Enrichment and KEGG Analysis
The protein-protein interaction (PPI) network for DE-miRNAs' predicted target genes was identified using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) online database (http://stringdb.org/) (accessed on 4 March 2022) [26]. Gene Ontology (GO) functional annotation for biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed for integrated target genes of DE-miRNAs. A p-value of <0.05 was regarded as statistically significant.

Identification and Analysis of Hub Gene
The protein-protein interaction (PPI) for DE-miRNAs from STRING database was exported to Cytoscape software (version 3.9) and visualized [27]. The top 30 hub genes were selected (the top 30 nodes of the PPI network) using the Maximal Clique Centrality (MCC) method [28], which has a better performance in terms of its precision in predicting the top essential proteins. Further analysis was performed using ClueGO [29] to integrate GO terms as well as KEGG pathways and create a functionally nested or organized GO/pathway term (k score = 3). This task analyzes one set of genes or compares two lists of genes and comprehensively visualizes functionally grouped terms [29].

Real-Time Polymerase Chain Reaction for Determination of mRNA Expression of Hub Genes
Total RNA extraction and complementary DNA synthesis were performed as previously described [30]. Briefly, testis samples for each animal were used to extract RNA by TRizol Invitrogen, Carlsbad, CA, USA) tissue homogenization method. The RNA concentration and quality were determined using a NanoDrop-1000 spectrophotometer (Thermo Scientific, Rockford, IL, USA), and all RNA samples were treated with DNAse I (Invitrogen) to remove the DNA contaminant. Complementary DNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) from each biological replicate and stored at −20 • C.
Statistical analysis was performed using SAS Analytics software (9.4 version; SAS Institute, Cary, NC, USA). A p value ≤ 0.05 is considered as statistically significant. Analysis of variance (ANOVA) was used to calculate statistical significance. All mRNA data are expressed as mean ± SEM. The correlation (r) between miRNA and mRNA was calculated using PROC CORR of SAS (Pearson correlation coefficient).

Protein Immunoblots
Western blots for each testis tissue sample from mature and immature dogs were performed separately by methods described previously [30]. Briefly, protein extraction methods included: the addition of protease and phosphatase inhibitor to the testis sample, homogenization, lysate incubation at 4 • C for 45 min, centrifugation (at 12,000× g for 20 min), and determination of protein concentrations. Protein lysates (60 µg/lane) were then electrophoresed through 12% SDS-PAGE gel (Bio-Rad Laboratories, Philadelphia, PA, USA) and then transferred onto a PVDF membrane (Bio-Rad Laboratories). Samples were incubated in 10% goat serum in PBS to block non-specific binding. After overnight incubation at 4 • C with primary antibodies [mouse monoclonal to DNMT1 (Catalog # MA5-16169) and rabbit polyclonal to PTEN (Catalog # 600-401-859) from Thermo Fisher Scientific, Waltham, MA, USA; and mouse polyclonal to actin (sc-47778) from Santa Cruz Biotechnology, Santa Cruz, CA, USA], membranes were washed in buffer containing 2% animal serum and 0.1% detergent. The membranes were then incubated in secondary antibodies [goat anti-mouse IgG-FITC for DNMTA1 and β-actin (sc-2010; Santa Cruz Biotechnology), and goat anti-rabbit IgG-FITC for PTEN (sc-2012; Santa Cruz Biotechnology) for 1 h at room temperature. The blots were then washed and scanned using the Pharos FX Plus system (Bio-Rad Laboratories). FITC fluorophore was excited at 488 nm and read at the emission wavelength of 530 nm. All possible negative controls, equivalent concentrations of nonspecific IgG or normal serum in place of the primary antibody, were included.

Results
For MiRNA-associated gene quantitative profiling, 32 upregulated and 12 downregulated miRNAs were included for in-silico analysis ( Figure 1). Similarities of the nucleotide sequences of differentially expressed canine miRNAs were comparable with those of homo sapiens (Table 3). These up-and down-regulated miRNAs were submitted to elucidate predicted genes. Of 32 upregulated miRNAs submitted, 31 predicted 560 genes (Supplementary file S2) and of 12 downregulated miRNAs, 11 predicted 53 genes (Supplementary file S3).
antibodies [goat anti-mouse IgG-FITC for DNMTA1 and β-actin (sc-2010; Santa Cruz Biotechnology), and goat anti-rabbit IgG-FITC for PTEN (sc-2012; Santa Cruz Biotechnology) for 1 h at room temperature. The blots were then washed and scanned using the Pharos FX Plus system (Bio-Rad Laboratories). FITC fluorophore was excited at 488 nm and read at the emission wavelength of 530 nm. All possible negative controls, equivalent concentrations of nonspecific IgG or normal serum in place of the primary antibody, were included.

Results
For MiRNA-associated gene quantitative profiling, 32 upregulated and 12 downregulated miRNAs were included for in-silico analysis ( Figure 1). Similarities of the nucleotide sequences of differentially expressed canine miRNAs were comparable with those of homo sapiens (Table 3). These up-and down-regulated miRNAs were submitted to elucidate predicted genes. Of 32 upregulated miRNAs submitted, 31 predicted 560 genes (Supplementary file S2) and of 12 downregulated miRNAs, 11 predicted 53 genes (Supplementary file S3).
The top-ranked 30 hub genes using Maximal Clique Centrality (MCC) method for upregulated miRNAs and downregulated miRNAs were screened and presented in Figure 3A and 3B, respectively. Table 4 shows the hub genes and their roles, tissue expression, and protein-protein interactions (up to six closely related genes), for up-and down-regulated miRNAs in adult dog testis. Canine miRNAs and associated hub genes are presented in Table 5. Further, the top 30 upregulated and downregulated hub genes' associated KEGG pathways are presented in Table 6.
To interpret functionally nested gene ontology and pathway annotation networks for the predicted genes of adult dog testis, ClueGo nested network analysis was performed, and the results are presented in Supplementary files S6 and S7 for up-(29 GO term groups and 23 KEGG pathway groups) and down-regulated miRNAs (8 GO term groups and 2 KEGG pathway groups), respectively. For up-regulated hub genes, a functionally grouped network with terms as nodes ( Figure 4A), GO-pathway terms specific for genes ( Figure 4B), and a chart with functional groups including specific terms ( Figure 4C) is presented. Similarly, for down-regulated hub genes, functionally grouped networks with terms as Animals 2023, 13, 1520 9 of 31 nodes ( Figure 5A), GO-pathway terms specific for genes ( Figure 5B), and a chart with functional groups including specific terms ( Figure 5C) is presented. terms as nodes ( Figure 5A), GO-pathway terms specific for genes ( Figure 5B), and a chart with functional groups including specific terms ( Figure 5C) is presented.
(A)         The mRNA expressions for CDKN1A, EGFR, JUN, NOTCH1, and PIK3R1 were greater (p < 0.05) in abundance in mature compared to immature dog testis (p < 0.05; Figure 6); whereas the mRNA expressions for DNMT1, PTEN, ESR1, and TIMP3 were lower in abundances in mature compared to the immature testis (p < 0.05; Figure 6). The mRNA expressions of hub genes for upregulated miRNA were in greater abundance and the mRNA expressions of hub genes for downregulated miRNA were in lower abundance (p < 0.05). There was a positive association for the miRNA-mRNA pair (Figure 7; r = 0.60; p < 0.05). The relative expressions of miRNA and associated hub gene mRNA in mature dog testis were also provided in Table 7.

Discussion
In the present study using DE-miRNAs between immature and adult dog testis samples, we identified 613 genes involved in the regulation of testis development. The GO biological functional enrichment showed that genes were mainly enriched in regulation of cellular process, cellular response to stimulus, developmental process, cell population proliferation, cell death, cell differentiation, apoptotic process, and cell metabolic process. The KEGG pathway enrichment showed that genes were mainly enriched in cancer biology, PI3K-Akt signaling pathway, AGE-RAGE signaling pathway, p53 signaling pathway, cellular senescence, hormone signaling pathway and human papillomavirus infection. Furthermore, we performed bioinformatics analysis to identify the potential key genes based on random selection algorithm, GO semantic similarity, PPI network, and cluster analysis. The results showed the involvement of differentially expressed genes in growth, sexual development, the maintenance of gluconeogenesis and lipid metabolism, cell proliferation, Sertoli and spermatogonial stem cells division and growth, cell cycle (cell cycle progression at G1-S phase), maturation, cell survival, and apoptosis. These key genes functioned as the essential molecules that might have mediated the testis development process between the immature and adult dogs.

Discussion
In the present study using DE-miRNAs between immature and adult dog testis samples, we identified 613 genes involved in the regulation of testis development. The GO biological functional enrichment showed that genes were mainly enriched in regulation of cellular process, cellular response to stimulus, developmental process, cell population proliferation, cell death, cell differentiation, apoptotic process, and cell metabolic process. The KEGG pathway enrichment showed that genes were mainly enriched in cancer biology, PI3K-Akt signaling pathway, AGE-RAGE signaling pathway, p53 signaling pathway, cellular senescence, hormone signaling pathway and human papillomavirus infection. Furthermore, we performed bioinformatics analysis to identify the potential key genes based on random selection algorithm, GO semantic similarity, PPI network, and cluster analysis. The results showed the involvement of differentially expressed genes in growth, sexual development, the maintenance of gluconeogenesis and lipid metabolism, cell proliferation, Sertoli and spermatogonial stem cells division and growth, cell cycle (cell cycle progression at G1-S phase), maturation, cell survival, and apoptosis. These key genes functioned as the essential molecules that might have mediated the testis development process between the immature and adult dogs.
Micro RNAs critically regulate the proliferation and/or early differentiation of stem cell populations in testis [31]. In mouse, deletion of both miR-34b and miR-34c led to sterility, resulting in reduced sperm count, changed sperm morphology, and abnormal motility [32]. It has been cited that miR-34c-5p could be used as biomarkers of germ cell maturation [33][34][35]. The differences in expressions of cfa-miR-34b and cfa-miR-34c were vast between mature and immature testis in the current investigation. The cfa-miR-34b and cfa-miR-34c associated with hub genes NOTCH1 and MYC regulates the cell cycle, cellular fate determination, cell proliferation, cell differentiation, and cellular apoptosis. MicroRNA-34b expression was associated with meiotic-specific cells from the murine testis [36] and miR-34c was highly expressed in pachytene spermatocytes and round spermatids when compared with testicular somatic cells and other tissues in adult mice [37]. In humans, miR-34b was downregulated in asthenozoospermic and oligoasthenozoospermic sperm compared with normal sperm, suggesting its function is critical beyond sperm production [38]. The enhanced expression of miR-34c in the germ cells during the later steps of spermatogenesis indicates its functional significance in meiosis and spermiogenesis. Retinoic acid receptor gamma is one of the target genes of miR-34c, indicating the critical role of retinoic acid signaling in the control of meiosis [39]. MicroRNA-34 is intrinsically linked to the p53 tumor suppressor gene and the established Wnt cascade [40]. Wnt/bcatenin signaling is essential for the regulation of spermatogenesis [41], and the gene p53 is important for the biogenesis of acrosome and nuclear shaping during spermiogenesis. Previous studies demonstrated that Sertoli proliferation and differentiation can be mediated through the Wnt/β-catenin signaling pathway [42,43], mTOR signaling pathway [44][45][46], and TGF-β signaling pathway [47,48]. PTEN, PI3K/AKT, and STAT signaling pathways were found to be involved in bull sperm cell apoptosis [49]. Recent study on analysis of miRNAs and target mRNAs between immature and mature bull testis showed enrichment during Sertoli proliferation and differentiation, and sperm apoptosis [50]. Further, several differentially expressed genes enriched in metabolic pathways were involved in fat metabolism, including fatty acid degradation, adipocytokine signaling, and PPAR signaling pathway [50].
In the current study, cfa-miR-7a was upregulated in adult testis and EGF was identified as one of its target genes. Further, this upregulated miRNA in adult testis-associated hub genes CDH1 and MET interacted with EGF and EGFR. The ErbB signaling pathwayassociated genes found were CDKN1A, EGFR, JUN, KRAS, MYC, and PIK3R1. The CDKN1A is the primary p53 target gene that mediates cell-cycle arrest [51,52]. LH signaling was reduced in CDKNIA knockout mice plausibly affecting pubertal development [53]. The EGF mediates spermatogonial proliferation through its receptors ErbB1, ErbB2, and ErbB4 in the testis [54]. It is possible that EGF mediates spermatogonial proliferation through its receptors on Sertoli cells via activation of MAPK cascade and/or PI3K cascade by elevating the expressions of SCF, Ig-NRG1, and EGFRs (ERBBRs) [55,56]. Further, KRAS expression patterns showed preferential tissue activation suggesting different cellular functions [57,58]. Interestingly, cfa-miR-125a was downregulated in adult testis and its associated hub genes were ERBB2 and ERBB3. Aberrant EGFR activation is a significant factor in the development and progression of multiple cancers [59] suggesting that a balanced expression is warranted in testis development.
In the current study, the upregulated cfa-miR-29c-associated gene was PIK3R1, and its signals are important for cell activities, including cell growth and division, migration, production of new proteins, transport of materials within cells, and cell survival. Studies suggest that PI3K signaling may be involved in the regulation of several hormones, including insulin. NF-kappaB and PI3K-Akt pathways are among PI3 K-associated pathways. PI3K-Akt pathway is an intracellular signal transduction pathway that promotes metabolism, proliferation, cell survival, growth, and angiogenesis in response to extracellular signals [60]. This is mediated through serine and/or threonine phosphorylation of a range of downstream substrates. The PI3K-Akt pathway engages in many stages of male reproduction, including the regulation of the hypothalamus-pituitary-gonad axis during spermatogenesis, the proliferation and differentiation of spermatogonia and somatic cells, and the regulation of sperm autophagy and testicular endocrine function in the presence of endocrine disrupting chemicals [61]. Further, the PI3K-Akt pathway is required for the stimulatory actions of FSH [62]. It should be noted that the activation of PI3K by EGF occurs via the association of the p85 subunit of PI3K (PIK3R1) with the activated EGFR [63]. PIK3R1/p85a is the most abundant isoform in normal tissues [64] but its expression is reduced in cancer suggesting that it regulates cell proliferation. In the current study, cluster analysis revealed PI3K-Akt pathway was regulated by associated genes BCL2L11, CCND1, CCNE1, CDK4, CDK6, CDKN1A, EGFR, FGFR1, JAK2, KDR, KRAS, MCL1, MET, MYC, PIK3R1, PTEN, and RELA.
Current investigation revealed that upregulated canine miR-15a and miR-378 were associated with hub gene VEGFA, which regulates neovascularization and cord formation, and potentially acts through the PI3K pathway during testis morphogenesis [65]. Further, VEGF signaling regulates germ cell proliferation and promotes testicular regeneration via direct action on germ cells and the enhancement of vascularization. Associated genes KDR, KRAS, and PIK3R1 in the VEGF signaling pathway in the current study, may regulate the testis morphogenesis and regeneration.
Regulation of testis development by hormones has been described in [66]. Cluster analysis in the current investigation revealed the involvement of associated genes EGFR, JUN, KRAS, and MMP2 in the regulation of the GnRH signaling pathway. The FSH regulates Sertoli cell proliferation during fetal and early postnatal life by activating cAMP/PKA/ERK1/2 and PI3K/Akt/mTORC1 dependent pathways, and by increasing the transcriptional activity of c-MYC and HIF2 and the expression of CCND1 [67]. The CCND1 is a hub gene for upregulated canine miRNAs 15a, 16, 19a and 20a, and MYC is a hub gene for upregulated miR-34c in mature testis in the current investigation, suggesting the involvement in the Sertoli cell proliferation as indicated in the previous studies. Insulin and IGF1 regulate testicular functions by activating PI3K/Akt and ERK1/2 signaling pathways. Mice lacking INSR and IGF1R in Sertoli cells showed a 72% reduction in testis size and a 79% reduction in daily sperm production [68,69]. Relaxin is another member of the insulin-related peptide family involved in Sertoli cell proliferation [66,70]. Associated genes EGFR, JUN, KRAS, MMP2, PIK3R1, and RELA engage in the relaxin signaling pathway in the current study, could have contributed to Sertoli cell proliferation. Genes IGF1, AMH, hedgehog (DHH), and platelet-derived growth factor (PDGF) seem to regulate Leydig cell differentiation and function. The IGF1 stimulated differentiation and mitosis of Leydig cells [71]. Equally, the decrease in estrogen production inhibited Leydig cell differentiation in prepubertal and adult rat testes [72]. Since IGF1, DHH, and PDGF are Sertoli cells paracrine factors, it seems reasonable to speculate that thyroid hormone actions on Leydig cells might be, at least in part, mediated through Sertoli cells. Relaxin-induced Sertoli cell proliferation involves the activation of a Gi protein and the activation of EKR1/2 and PI3K/Akt pathways [66,70]. Activin A along with FSH regulates Sertoli cell proliferation during the fetal and postnatal period via the SMAD pathway [73,74]. The NOTCH1 is identified as a hub gene for upregulated cfa-miR-34c in the current investigation. NOTCH1-IC forms a transcriptional complex with SMAD, a component of established TGF-β signaling, and regulates the expression of HES1 by binding to the promoter [75]. These signal cross-talks are sophisticated regulation processes during cell fate determination and cancer development [76]. Interestingly, Nemo-like kinase (NLK) was a hub gene for downregulated miR-181a, -181b. The NLK phosphorylates the NOTCH1 protein. NLK-mediated phosphorylation does not interfere with the nuclear localization of NOTCH1-IC but decreases the association of the Notch active transcription complex [77]. Inhibin B is the main circulating inhibin produced by Sertoli cells; however, inhibin B has no role by itself but plays a role in the modulation of activin A-induced Sertoli cell proliferation [66,67].
Cytokines play an important regulatory role in the development and normal function of the testis. They signal via the adapter protein MyD88 to activate NFκB; and TGFβs and activins, which signal through serine/threonine kinase receptor subunits to activate SMAD transcription factors. It has been shown that IL1α and IL1β increased DNA synthesis and Sertoli cell number in vitro and IL1α had a more potent effect than IL1β [78]. TNFR1 has been detected in Sertoli cells and probably mediates TNFα biological actions, proinflammatory and immunoregulatory responses, and apoptosis [79]. A chemokine, CXCL8, was identified as one of top 30 hub genes in the current study. It should be noted that PTEN (hub gene for upregulated miR-22 and downregulated miR-214) loss induces a selective upregulation of CXCL8 signaling that sustains cell growth and survival [80]. In the current study, cluster analysis revealed involvement of T and B cell receptor signaling pathway regulated by CDK4, JUN, KRAS, PIK3R1, and RELA genes. This regulation could have contributed to the development and normal function of the testis.
Androgen-dependent regulation of Sertoli cell proliferation is an indirect effect probably exerted through the secretion of a paracrine factor [66,67]. Direct effects of androgens on Sertoli cells seem to be related to maturation of this cell type. Estrogens play important roles in the regulation of testis development and spermatogenesis. Associated genes EGFR, ESR1, JUN, KRAS, MMP2, and PIK3R1 were involved in the estrogen signaling pathway in the current study. ESR was a hub gene for miR-18a. -19a, and -22 in the current study. Estrogens increase proliferation of Sertoli cells through ERα and GPER and on the other hand, at the end of the proliferative period it promotes cessation of proliferation and cell maturation through ERβ [66,67]. ERα promotes cell proliferation through the activation of NFκB in a PI3Kand a ERK1/2-dependent manner and that this is accompanied by CCND1 induction [81]. GPER activates Src/PI3K/Akt pathway which participates in E2-induced Sertoli cell proliferation via regulating the expression of S-phase kinase-associated protein 2 (SKP2) [82]. The ERβ promotes cell cycle exit and cell maturation through the activation of CREB in a PI3K-dependent manner and this leads to the expression of the Sertoli cell differentiation markers-CDKN1B, GATA1 (isoform GATA6 was a hub gene for downregulated cfa-miR-181a and -181b), and DMRT1 [66,67]. Role of thyroid hormone and retinoic acid in the cessation of proliferation and in maturation of Sertoli cells. Thyroid hormone regulated Sertoli cell maturation is through TRα1 and AIMP1 (p43) receptors. The mechanisms participating in these processes involve the regulation of CX43, c-MYC (MYC was identified as a hub gene for upregulated cfa-miR-34b and MYCN a hub gene for miR-101), P21CIP1 (CDKN1A a hub gene for upregulated cfa-miR-106a), and P27KIP1 (CDKN1B a hub gene for down-regulated cfa-miR-181a) [83][84][85]. Cluster analysis revealed associated genes CCND1, ESR1, FOXO1, KRAS, MYC, NOTCH1, and PIK3R1 involved in the regulation of the thyroid signaling pathway.
Studies observed positive (upregulated-upregulated; complement) or negative (upregulated-downregulated; reverse complement) associations between miRNA and mRNA pairs [86]. It should be noted that miRNAs can activate gene expression directly or indirectly in response to different cell types and environments and in the presence of distinct cofactors. The biological outcome of miRNA-mRNA interaction can be altered by several factors contributing to the binding strength and repressive effect of a potential target site. Interestingly, it has been reported that significant miRNA-mRNA associations were complementing in normal tissue and miRNA-mRNA whereas associations were reverse complementing in the same tissue in diseased conditions [87]. The association between miRNA and hub gene mRNA expressions was positive in the current study.
Collectively, in the current study, GO analysis of differentially expressed genes showed that the down-regulated genes were significantly enriched in a large number of GO terms. Further, the GO analysis showed that most GO terms were downregulated, indicating that these DEGs may have played important roles in the development of the testis of immature dogs. However, the upregulated genes enriched in the GO terms were related to meiosis, protein ubiquitination, and fertilization of adult dogs. This suggests that a large number of genes with key roles in male reproduction traits are highly expressed in mature testis. Interestingly these genes are not expressed or expressed in low abundance in immature dog testis. Similar results were reported in a bull study [50]. Our analysis identified differentially expressed genes and differentially expressed miRNAs associated with male reproduction and elaborated cluster networks between miRNAs and genes regulating testis structure and function. This outcome provides significant insights into the molecular mechanisms of male fertility and spermatogenesis and will be valuable for future genetic and epigenetic studies of testis development and maturity.

Conclusions
The complex relationship between miRNA and gene interaction is a vital component of miRNA functional analysis in the testis development process. Abnormal expression of miRNAs and/or any regulation disturbance can lead to impaired germ cells, abnormal spermatogenesis, and even neoplasia. The present in-silico analysis showed the involvement of canine testicular miRNAs in structure and function. The DE-miRNAs between immature and adult canine testis and their associated genes involved in several regulatory pathways play a crucial role during the immature testis transition to the adult testis. A focused study of individual miRNA molecules may elucidate specific functions or problems, endorse the development of anti-oncogenic reagents and infertility/subfertility treatments, and bolster novel contraceptive technologies.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ani13091520/s1, Supplementary file S1: The ethidium bromidestained electrophoresis gel, with amplicons of expected sizes. Supplementary file S2: Upregulated miRNAs-and miRNet-based target and predicted genes in cows with metritis. Supplementary file S3: Downregulated miRNAs-and miRNet-based target and predicted genes in cows with metritis. Supplementary file S4: STRING-based protein-protein interaction network predicted biological processes, cellular components, molecular functions, and KEGG pathways for upregulated miRs predicted genes. Supplementary file S5: STRING-based protein-protein interaction network predicted biological processes, cellular components, molecular functions, and KEGG pathways for downregulated miRs predicted genes. Supplementary file S6: ClueGo-based protein-protein interaction predicted biological processes, cellular components, molecular functions, and KEGG pathways for upregulated miRNAs predicted hub genes. Supplementary file S7: ClueGo-based protein-protein interaction predicted biological processes, cellular components, molecular functions, and KEGG pathways for downregulated miRNAs predicted hub genes. Supplementary file S8: Representative Western blots of isozymes PTEN, phosphatase and tensin homolog; DNMT1, DNA methyltransferase 1; and ACTB, beta actin.