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Background: Testicular germ cell tumours (TGCTs) represent a clinical challenge; they are most prevalent in young individuals and are triggered by molecular mechanisms that are not fully understood. The origin of TGCTs can be traced back to primordial germ cells that fail to mature during embryonic development. These cells express high levels of pluripotency factors, including the transcription factor NANOG which is highly expressed in TGCTs. Gain or amplification of the NANOG locus is common in advanced tumours, suggesting a key role for this master regulator of pluripotency in TGCT stemness and malignancy. Methods: In this study, we analysed the expression of microRNAs (miRNAs) that are regulated by NANOG in TGCTs via integrated bioinformatic analyses of data from The Cancer Genome Atlas and NANOG chromatin immunoprecipitation in human embryonic stem cells. Through gain-of-function experiments, MIR9-2 was further investigated as a novel tumour suppressor regulated by NANOG. After transfection with MIR9-2 mimics, TGCT cells were analysed for cell proliferation, invasion, sensitivity to cisplatin, and gene expression signatures by RNA sequencing. Results: For the first time, we identified 86 miRNAs regulated by NANOG in TGCTs. Among these, 37 miRNAs were differentially expressed in NANOG-high tumours, and they clustered TGCTs according to their subtypes. Binding of NANOG within 2 kb upstream of the MIR9-2 locus was associated with a negative regulation. Low expression of MIR9-2 was associated with tumour progression and MIR9-2-5p was found to play a role in the control of tumour stemness. A gain of function of MIR9-2-5p was associated with reduced proliferation, invasion, and sensitivity to cisplatin in both embryonal carcinoma and seminoma tumours. MIR9-2-5p expression in TGCT cells significantly reduced the expression of genes regulating pluripotency and cell division, consistent with its functional effect on reducing cancer stemness. Conclusions: This study provides new molecular insights into the role of NANOG as a key determinant of pluripotency in TGCTs through the regulation of MIR9-2-5p, a novel epigenetic modulator of cancer stemness. Our data also highlight the potential negative feedback mediated by MIR9-2-5p on NANOG expression, which could be exploited as a therapeutic strategy for the treatment of TGCTs. [ABSTRACT FROM AUTHOR]