Silencing of LINC00467 inhibits cell proliferation in testicular germ cell tumors cells

A significant decrease in LINC00467 expression in testicular germ cell tumors (TGCTs) was found in our previous study in comparison to adjacent tissue. Interestingly, the expression of LINC00467 correlated with the pathological grade of the tumor in TGCT patients. The higher the expression of LINC00467 was, the worse the prognosis of the patients with TGCT was. Despite these findings, the exact role of LINC00467 in the development of TGCTs requires further investigation. LINC00467 expression was downregulated in the NCCIT and TCam-2 cell lines via small interfering RNA (siRNA) silencing. The levels of gene expression were validated using quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Cell proliferation was evaluated by the MTT and Cell Counting Kit-8 (CCK8) assays, whereas flow cytometry was used to assess the effects on the cell cycle. Western blotting analysis was used to detect expression levels of protein. Additionally, RNA-sequencing and bioinformatics methods were used to investigate the mechanism of action of LINC00467 in TGCTs. The suppression of LINC00467 expression resulted in decreased cell proliferation and induced S-phase arrest. Furthermore, the suppression of LINC00467 downregulated proliferating cell nuclear antigen (PCNA), a protein related to cell cycle regulation, while it upregulated p21 expression. In other studies involving dihydrotestosterone (DHT) stimulation, it was observed that DHT could upregulate LINC00467 expression. In addition, silencing of the LINC00467 reversed the effect of testosterone on cell proliferation. The Gene Set Enrichment Analysis (GSEA) revealed that LINC00467 regulated the p53 pathway by modulating the expression of CCNG1. Our study found that LINC00467 regulates cell proliferation by inducing S-phase arrest through the cell cycle-related proteins PCNA and p21. These findings contribute to our understanding of non-coding RNAs mechanisms involved in the development of TGCTs

MAGEB2-Mediated Degradation of EGR1 Regulates the Proliferation and Apoptosis of Human Spermatogonial Stem Cell Lines

Spermatogonial stem cells are committed to initiating and maintaining male spermatogenesis, which is the foundation of male fertility. Understanding the mechanisms underlying SSC fate decisions is critical for controlling spermatogenesis and male fertility. However, the key molecules and mechanisms responsible for regulating human SSC development are not clearly understood. Here, we analyzed normal human testis single-cell sequencing data from the GEO dataset (GSE149512 and GSE112013). Melanoma antigen gene B2 (MAGEB2) was found to be predominantly expressed in human SSCs and further validated by immunohistology. Overexpression of MAGEB2 in SSC lines severely weakened cell proliferation and promoted apoptosis. Further, using protein interaction prediction, molecular docking, and immunoprecipitation, we found that MAGEB2 interacted with early growth response protein 1 (EGR1) in SSC lines. Reexpression of EGR1 in MAGEB2 overexpression cells partially rescued decreased cell proliferation. Furthermore, MAGEB2 was shown to be downregulated in specific NOA patients, implying that abnormal expression of MAGEB2 may impair spermatogenesis and male fertility. Our results offer new insights into the functional and regulatory mechanisms in MAGEB2-mediated human SSC line proliferation and apoptosis

PPARγ maintains the metabolic heterogeneity and homeostasis of renal tubulesResearch in context

Background: The renal tubules, which have distant metabolic features and functions in different segments, reabsorb >99% of approximately 180 l of water and 25,000 mmol of Na + daily. Defective metabolism in renal tubules is involved in the pathobiology of kidney diseases. However, the mechanisms underlying the metabolic regulation in renal tubules remain to be defined. Methods: We quantitatively compared the proteomes of the isolated proximal tubules (PT) and distal tubules (DT) from C57BL/6 mouse using tandem mass tag (TMT) labeling-based quantitative mass spectrometry. Bioinformatics analysis of the differentially expressed proteins revealed the significant differences between PT and DT in metabolism pathway. We also performed in vitro and in vivo assays to investigate the molecular mechanism underlying the distant metabolic features in PT and DT. Findings: We demonstrate that the renal proximal tubule (PT) has high expression of lipid metabolism enzymes, which is transcriptionally upregulated by abundantly expressed PPARα/γ. In contrast, the renal distal tubule (DT) has elevated glycolytic enzyme expression, which is mediated by highly expressed c-Myc. Importantly, PPARγ transcriptionally enhances the protease iRhom2 expression in PT, which suppresses EGF expression and secretion and subsequent EGFR-dependent glycolytic gene expression and glycolysis. PPARγ inhibition reduces iRhom2 expression and increases EGF and GLUT1 expression in PT in mice, resulting in renal tubule hypertrophy, tubulointerstitial fibrosis and damaged kidney functions, which are rescued by 2-deoxy-d-glucose treatment. Interpretation: These findings delineate instrumental mechanisms underlying the active lipid metabolism and suppressed glycolysis in PT and active glycolysis in DT and reveal critical roles for PPARs and c-Myc in maintaining renal metabolic homeostasis. FUND: This work was supported by the National Natural Science Foundation of China (grants 81572076 and 81873932; to Q.Z.), the Applied Development Program of the Science and Technology Committee of Chongqing (cstc2014yykfB10003; Q.Z.), the Program of Populace Creativities Workshops of the Science and Technology Committee of Chongqing (Q.Z.), the special demonstration programs for innovation and application of techniques (cstc2018jscx-mszdX0022) from the Science and Technology Committee of Chongqing (Q.Z.). Keywords: PPARγ, PPARα, C-Myc, Nrf2, Lipid metabolism, Glycolysis, Renal proximal tubules, Renal distal tubules, Kidne

PPP3CB Inhibits Migration of G401 Cells via Regulating Epithelial-to-Mesenchymal Transition and Promotes G401 Cells Growth

PPP3CB belongs to the phosphoprotein phosphatases (PPPs) group. Although the majority of the PPP family play important roles in the epithelial-to-mesenchymal transition (EMT) of tumor cells, little is known about the function of PPP3CB in the EMT process. Here, we found PPP3CB had high expression in kidney mesenchymal-like cells compared with kidney epithelial-like cells. Knock-down of PPP3CB downregulated epithelial marker E-cadherin and upregulated mesenchymal marker Vimentin, promoting the transition of cell states from epithelial to mesenchymal and reorganizing the actin cytoskeleton which contributed to cell migration. Conversely, overexpression of PPP3CB reversed EMT and inhibited migration of tumor cells. Besides, in vitro and in vivo experiments indicated that the loss of PPP3CB suppressed the tumor growth. However, the deletion of the phosphatase domain of PPP3CB showed no effect on the expression of E-cadherin, migration, and G401 cell proliferation. Together, we demonstrate that PPP3CB inhibits G401 cell migration through regulating EMT and promotes cell proliferation, which are both associated with the phosphatase activity of PPP3CB

MiR-30a Inhibits the Epithelial—Mesenchymal Transition of Podocytes through Downregulation of NFATc3

MicroRNAs (miRNAs) possess an important regulating effect among numerous renal diseases, while their functions in the process of epithelial-to-mesenchymal transition (EMT) after podocyte injury remain unclear. The purpose of our study is to identify the potential functions of miR-30a in EMT of podocytes and explore the underlying mechanisms of miR-30a in the impaired podocytes. The results revealed that downregulation of miR-30a in podocyte injury animal models and patients, highly induced the mesenchymal markers of EMT including Collagen I, Fibronectin and Snail. Furthermore, overexpression of miR-30a enhances epithelial markers (E-cadherin) but diminished mesenchymal markers (Collagen I, Fibronectin and Snail) in podocytes. In addition, we established miR-30a target NFATc3, an important transcription factor of Non-canonical Wnt signaling pathway. More importantly, our findings demonstrated that the augmentation of miR-30a level in podocytes inhibits the nuclear translocation of NFATc3 to protect cytoskeleton disorder or rearrangement. In summary, we uncovered the protective function of miR30a targeting NFATc3 in the regulation of podocyte injury response to EMT

MiR542-3p Regulates the Epithelial-Mesenchymal Transition by Directly Targeting BMP7 in NRK52e

Accumulating evidence demonstrated that miRNAs are highly involved in kidney fibrosis and Epithelial-Eesenchymal Transition (EMT), however, the mechanisms of miRNAs in kidney fibrosis are poorly understood. In this work, we identified that miR542-3p could promote EMT through down-regulating bone morphogenetic protein 7 (BMP7) expression by targeting BMP7 3′UTR. Firstly, real-time PCR results showed that miR542-3p was significantly up-regulated in kidney fibrosis in vitro and in vivo. Moreover, Western blot results demonstrated that miR542-3p may promote EMT in the NRK52e cell line. In addition, we confirmed that BMP7, which played a crucial role in anti-kidney fibrosis and suppressed the progression of EMT, was a target of miR542-3p through Dual-Luciferase reporter assay, as did Western blot analysis. The effects of miR542-3p on regulating EMT could also be suppressed by transiently overexpressing BMP7 in NRK52e cells. Taken together, miR542-3p may be a critical mediator of the induction of EMT via directly targeting BMP7

Single-cell multi-omics analysis of human testicular germ cell tumor reveals its molecular features and microenvironment

Abstract Seminoma is the most common malignant solid tumor in 14 to 44 year-old men. However, its molecular features and tumor microenvironment (TME) is largely unexplored. Here, we perform a series of studies via genomics profiling (single cell multi-omics and spatial transcriptomics) and functional examination using seminoma samples and a seminoma cell line. We identify key gene expression programs share between seminoma and primordial germ cells, and further characterize the functions of TFAP2C in promoting tumor invasion and migration. We also identify 15 immune cell subtypes in TME, and find that subtypes with exhaustion features were located closer to the tumor region through combined spatial transcriptome analysis. Furthermore, we identify key pathways and genes that may facilitate seminoma disseminating beyond the seminiferous tubules. These findings advance our knowledge of seminoma tumorigenesis and produce a multi-omics atlas of in situ human seminoma microenvironment, which could help discover potential therapy targets for seminoma