miRNAs regulate genes post-translationally by binding to specific mRNA targets and thus participating in various physiological and pathological regulatory processes 12. Preliminary experiments have ascertained that miRNAs expression in spermatogenic cells changes at different stages, which indicates miRNAs contribute to spermatogenic cells development and promote spermatogenesis. Recent studies have confirmed that miRNAs, as key mediators of human and animal testicular development and spermatogenesis, are involved in early male germ cell proliferation and late spermatogenesis to form functional sperm 3. Since spermatogenesis is a complex differentiation process, NOA may be caused by abnormal gene expression at any stage during spermatogenesis 13. Accumulating evidence has confirmed that miR-188-3p is abundantly detected in spermatogonia and spermatocytes in human testicular tissue and during the meiotic cell cycle of mouse cells 7. Moreover, our previous research data supported miR-188-3p expression in NOA testicular tissue was significantly reduced, indicating the dysregulated miR-188-3p expression may contribute to the occurrence of NOA. In this experiment, a total of 109 differentially expressed mRNAs were identified by high-throughput sequencing analysis of testicular tissues of patients in the two groups. Among them, 5 mRNAs were predicted by bioinformatics analysis to be targets of miR-188-3p. The identified potential binding sites provided a new target for our subsequent study of the involvement of miR-188-3p in spermatogenesis. Given that previous studies reported that ATG7 is an autophagy gene and an E1-like activating enzyme that is essential for the autophagy coupling system and autophagosome formation. ATG7 can simultaneously control the number and size of autophagosomes 14. Later studies suggested that ATG7 is crucial for the formation of the ATG5/ATG12/ATG16L complex and LC3 complex and contributes to the regulation of the autophagy pathway 15. In animal experiments, Wang et al. found that male mice with ATG7 mutations led to complete infertility and abnormal acrosome formation. This was consistent with human round sperm disease, a severe fertility disorder characterized by abnormal round head sperm 16. Subsequently, it was found that the expression of ATG7 increased in males with infertility due to varicocele, and the level of autophagy and apoptosis increased, which may be related to the elimination of a large number of abnormal sperm and the response to heat and oxidative stress 17. Our results showed that ATG7 was considerably enhanced in NOA testicular samples and was expressed in spermatogenic cells at all levels, suggesting that ATG7 may participate in the development of NOA through high levels of autophagy.
miRNAs regulate genes expression during the post-transcriptional level through base pairing to cause translational inhibition and/or degradation of mRNA, thereby exerting their functions in biological and pathological processes 12. Previous studies have observed that the expression phenotype of testicular tissue in NOA patients is characterized by the loss of normal spermatogenesis, and the upregulated expression of some mRNA genes is caused by the downregulated expression of miRNA genes 18. In addition, Wang K et al confirmed that autophagy promoting factor (APF) regulates autophagy cell death by targeting miR-188-3p and ATG7 19. Previously, studies have shown that proteins related to chromosome structure (such as histones and dislocation repair proteins) may have binding sites with miR-188-3p, and these proteins are crucial for spermatogenesis 20. Afterwards, miR-188-3p was proven to be the target of polypyrimidine bundle binding protein (PTBP) 2 in mouse testes, and PTBP2 binds to the 3'-UTR of germ cell-specific phosphoglycerate kinase 2. Therefore, there may be another potential pathway by which miR-188-3p regulates PTBP2 involvement in spermatogenesis 21. Our previous study detected low expression of miR-188-3p in NOA testes. Furthermore, in this experiment, we detected high expression of the ATG7 gene in NOA testes. After miR-188-3p mimic transfected in the human testicular cancer cell line NTERA-2, the expression level of ATG7 was downregulated. In addition, after miR-188-3p inhibitor transfected, ATG7 expression was upregulated. The above results suggest ATG7 gene expression is impeded by miR-188-3p. However, it has not been reported whether miR-188-3p and ATG7 have a direct targeted regulatory relationship in testicular tissues to affect spermatogenesis. We further predicted through bioinformatics that the 3'-UTR of ATG7 have potential binding sites with miR-188-3p. Dual luciferase reporter assay results showed that after overexpression of miR-188-3p, the luciferase activity of wild-type ATG7 3’-UTR decreased, while the activity of mutant ATG7 3'-UTR showed no significant difference. Based on the results of these experiments, we speculate miR-188-3p may contribute to autophagy by regulating ATG7 expression, leading to spermatogenesis disorder and then NOA.
Autophagy is a relatively conservative evolutionary mechanism in eukaryotic cells. It begins with the formation of autophagosomes. Autophagy is important for normal cell development and physiological processes. In most cases, autophagy is recognized to protect and destroy cells 22. LC3 is a homologous gene of ATG8 in mammals 23 and has two mutually transformable forms, LC3-I and LC3-II. After induction of autophagy, LC3 is cleaved by the cysteine protease ATG4 to form LC3-I. LC3-I is coupled to the substrate phosphatidylethanolamine (PE) to form the autophagosomal membrane-bound form LC3-II. This process is performed by the E1-like enzyme ATG7, the E2-like enzyme ATG3, and the E3-like enzyme ATG5-. ATG12-ATG16L complex together. LC3-II, an important marker molecule for autophagosomes, increases with an increase in autophagosomal membranes 24. When autophagy is initiated, LC3-I will enzymatically cleave a small piece of polypeptide and transform into LC3-II. Therefore, the autophagy level can be estimated by the LC3-II/I ratio 25. Wei et al. found that the abnormal increase and decrease in LC3-II levels led to autophagy defects, thereby impairing the spermatogenic function of rat testes, leading to testicular developmental disorders 26. Beclin-1, as a homologue of ATG6 in mammals, is the core component of the autophagy mechanism 27. Beclin-1 is phosphorylated by unc-51-like autophagy activating kinase-1 (ULK1) and acts as an overall scaffold for the phosphoinositide 3-kinase (PI3K) complex, promoting proteins recruitment to autophagy vesicles. When the complex binds to other regulatory proteins, it selectively participates in different stages of autophagy 28. In addition, Beclin-1 has an independent mechanism of autophagy and an independent role in autophagy in cancer 29. In chronic myeloid leukaemia, miR-21 is upregulated. The expressions of Beclin-1, Vps34, and LC3-II are increased with treatment of anti-miR-21, which ultimately leads to increased autophagy 30. In this study, through immunohistochemistry, LC3 protein was found in the cytoplasm of spermatogenic cells at all levels. In addition, as the autophagy marker genes, the expression of LC3 and Beclin-1 in NOA testicular samples was higher than that in normal tissues. In patients with NOA, the expression of the two proteins was shown to increase through Western blot. Highly expressed LC3 and Beclin-1 may participate in the occurrence of NOA through autophagy.
miRNAs are involved in cellular autophagy expression and regulate the target genes expression at the chromatin organization, transcriptional and post-transcriptional levels. miRNAs target the 3' untranslated region of mRNAs, turn on/off the gene expression of key autophagy factors after transcription, and regulate the promotion of autophagy factors to trigger or inhibit the occurrence and development of cellular autophagy. It is known that the interaction between miR-471-5p and autophagy member protein regulates the clearance of apoptotic germ cells through LC3-related phagocytosis. Transgenic mice expressing miR-471-5p in Sertoli cells (SCs) showed increased germ cell apoptosis and impaired male fertility 31. In our study, autophagy marker genes expression in NOA testicular tissue was detected to be higher than that of the normal group by qRT-PCR and Western blot. Additionally, in cell experiments, after miR-188-3p overexpression, LC3 and Beclin-1 expression was lower. After miR-188-3p suppression, LC3 and Beclin-1 were enhanced in the unsuppressed individuals. The immunofluorescence results showed that after miR-188-3p overexpressed, LC3 spot aggregation elevated in comparison with that in the non-overexpression samples. After miR-188-3p expression was suppressed, LC3 spot aggregation decreased compared with that in the non-inhibition group. Finally, autophagy was further observed through transmission electron microscopy32. The number of autophagosomes in the miR-188-3p overexpression group was observed to be lower than that in the non-overexpression group through transmission electron microscopy. Our results indicated miR-188-3p is related with the occurrence of autophagy. In our study, first, the expression of ATG7 in testicular tissues of the NOA group and normal group was confirmed to be elevated by high-throughput sequencing. It has been proven that ATG7 contribute to autophagy. Wang et al. also confirmed that miR-188-3p impede autophagy by regulating ATG7 19. We further used dual luciferase experiments to verify that miR-188-3p have a mutual regulatory relationship with ATG7 and ATG7 is the target site for miR-188-3p. There may be downregulation of miR-188-3p in the testicular samples by reducing these two genes. The inhibitory effect of ATG7 in turn leads to an abnormal increase in autophagy levels, which affects the spermatogenesis process.
In summary, the results of this study indicate miR-188-3p may regulate its target gene ATG7 to participate in autophagy and thus affect the occurrence and development of NOA. This provides a new experimental and theoretical basis for studying autophagy as a potential target for diagnosis and treatment. Combined with our previous research results that miR-188-3p-targeted regulation of MLH1 affects apoptosis and participates in the occurrence of NOA, it will be possible to explore whether there is an autophagy-apoptosis interaction network affecting NOA.