Aerobic glycolysis is one of the characteristics of tumor metabolism and contributes to the development of tumors. Studies have identified that microRNA (miRNA/miR) serves an important role in glucose metabolism of tumors. miR‑199a‑3p is a member of the miR‑199a family that controls the outcomes of cell survival and death processes, and previous studies have indicated that the expression of miR‑199a‑3p is low and may be an inhibitor in several cancer types, including testicular tumors. The present study discussed the role and underlying mechanism of miR‑199a‑3p in aerobic glycolysis of Ntera‑2 cells and identified its downstream factors. Firstly, miR‑199a‑3p exhibited an inhibitory effect on lactic acid production, glucose intake, and reactive oxygen species (ROS) and adenosine 5'‑triphosphate (ATP) levels in Ntera‑2 cells. Then, using bioinformatics, recombinant construction and a dual luciferase reporter gene system, transcription factor Specificity protein 1 (Sp1) was determined as the direct target of miR‑199a‑3p. Also, downregulation of Sp1 by RNA interference decreased lactic acid production, glucose intake, and ROS and ATP levels in Ntera‑2 cells. Subsequently, through a functional rescue experiment, it was identified that the overexpression of Sp1 may abate the inhibition of miR‑199a‑3p on glucose metabolism, with the exception of ATP level, suggesting a reciprocal association between Sp1 and miR‑199a‑3p. Finally, it was determined that miR‑199a‑3p overexpression and Sp1 knockdown decreased lactate dehydrogenase A (LDHA) protein expression, which indicated that LDHA is a downstream target of the miR‑199a‑3p/Sp1 signaling pathway. To additionally verify the regulation of LDHA expression by 199a‑3p/Sp1, a LDHA promoter reporter plasmid was generated and the high activity of the promoter, which contained 3 potential Sp1 binding elements, was confirmed. In addition, the overexpression of Sp1 led to the increased activity of the LDHA promoter, whereas knockdown of Sp1 exhibited the opposite effect. Therefore, the results of the present study demonstrated that miR‑199a‑3p can inhibit LDHA expression by downregulating Sp1, and provided mechanistic evidence supporting the existence of a novel miR‑199a‑3p/Sp1/LDHA axis and its critical contribution to aerobic glycolysis in testicular cancer cells.

Testicular germ cell tumors (TGCTs) are a diverse group of neoplasms that are derived from dysfunctional fetal germ cells and can also present in extragonadal sites. The genetic drivers underlying malignant transformation of TGCTs have not been fully elucidated so far. The aim of the present study is to clarify the functional role and regulatory mechanism of miR‐196a‐5p in TGCTs. We demonstrated that miR‐196a‐5p was downregulated in TGCTs. It can inhibit the proliferation, migration, and invasion of testicular tumor cell lines including NT‐2 and NCCIT through targeting the NR6A1 gene, which we proved its role in promotion of cell proliferation and repression of cellular junction and aggregation. Mechanistically, NR6A1 inhibited E‐cadherin through binding with DR0 sites in the CDH1 gene promoter and recruiting methyltransferases Dnmt1. Further, NR6A1 promoted neuronal marker protein MAP2 expression in RA‐induced neurodifferentiation of NT‐2 cells and testicular tumor xenografts. Clinical histopathologically, NR6A1 was positively correlated with MAP2, and negatively correlated with E‐cadherin in TGCTs. These findings revealed that the miR‐196a‐5p represses cell proliferation, migration, invasion, and tumor neurogenesis by inhibition of NR6A1/E‐cadherin signaling axis, which may be a potential target for diagnosis and therapy of TGCTs.

Bacterial infection and induced inflammation are important causes of male infertility. Here, we described the characteristics of expression and the regulatory role of nuclear receptor subfamily 2 group C member 2 (NR2C2) in testicular inflammatory injury induced by infection with the bacterial endotoxin LPS. We found that NR2C2 was highly expressed in the testes and the expression of NR2C2 was upregulated in testicular macrophages in the LPS-induced mouse orchitis model in vivo. In primary testicular macrophages and RAW264.7 cells in vitro, RNA interference with the Nr2c2 gene downregulated the expression of inflammatory factors such as IL-1β and IL-6. In addition, the knockdown of NR2C2 in macrophages alleviated the inhibitory effect of the inflammatory supernatant secreted by the macrophages on the proliferation of spermatogonia GC-1 SPG cells. Mechanistically, NR2C2 activated NF-κB signaling by binding with DR elements in the promotor of the Nfκb gene and promoted the development of inflammation. These data are the first to confirm that during LPS-induced bacterial infection, NR2C2 plays a proinflammatory role by activating IL-1β and IL-6 via the NF-κB pathway in testicular macrophages, consequently inhibiting the proliferation of spermatogonia and damaging the quality of sperm. Our findings reveal the important role of NR2C2 in testicular inflammatory injury induced via LPS and provide a new potential target and a molecular basis for the treatment of male infertility caused by bacterial infection.

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