RNA N6-methyladenosine (m6A) readers mediate cancer progression. However, the functional role and potential mechanisms of the m6A readers in prostate cancer tumorigenicity remain to be elucidated. In this study, we demonstrate that YTHDF3 expression is elevated in castration-resistant prostate cancer (CRPC) and positively correlated to high grade, bone metastasis and poor survival. YTHDF3 expression promoted CRPC cell proliferation, epithelial to mesenchymal transition (EMT) and tumour progression. Mechanistically, YTHDF3 promoted the RNA degradation of SPOP and NXK3.1 but stabilized RNA expressions of TWIST1 and SNAI2 dependent on m6A to facilitate cell proliferation and EMT. Additionally, YTHDF3 expression enhanced AKT activity via degrading SPOP in an m6A-dependent manner. Importantly, we found that melatonin can compete with m6A to occupy the m6A-binding cage of YTHDF3, leading to inhibition of YTHFD3 and its target expressions as well as CRPC tumour growth. Our findings uncover an essential role of YTHDF3 in the progression of CRPC and highlight the role of melatonin in anti-CRPC activity.

BACKGROUND: Lung cancer is a common malignant tumor with high morbidity and mortality rate. Glucosamine 6-phosphate N-acetyltransferase (GNPNAT1), which serves as a critical enzyme in hexosamine biosynthetic pathway (HBP), has been identified as a metastasis-associated gene and is upregulated in lung adenocarcinoma (LUAD). However, the exact role and related mechanism of GNPNAT1 in LUAD metastasis remain unknown.
METHODS: We analyzed the expression of GNPNAT1 in the public databases and confirmed the results by immunohistochemistry (IHC). The biological functions of GNPNAT1 in LUAD were investigated based on The Cancer Genome Atlas (TCGA). Correlations between GNPNAT1 and cancer immune characteristics were analyzed via the Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) and Cell-type Identification by Estimating Relative Subsets of RNA Transcript (CIBERSORT) R package. The underlying mechanisms of altered GNPNAT1 expression on LUAD cell tumorigenesis, proliferation, migration, invasion, and metastasis were explored in vitro and in vivo.
RESULTS: We demonstrated that GNPNAT1 expression was significantly increased in LUAD and negatively associated with the overall survival (OS) of patients. hsa-miR-1-3p and hsa-miR-26a-5p were identified as upstream miRNA targets of GNPNAT1. GNPNAT1 was associated with the infiltration levels of CD8 T cells, memory-activated CD4 T cells, NK cells resting, macrophages M0, macrophages M1, neutrophils, gamma delta T cells, and eosinophils, while it was negatively correlated with memory-resting CD4 T cells, regulatory T cells (Tregs), resting NK cells, monocytes, resting dendritic cells, and resting mast cells. GNPNAT1 knockdown significantly inhibited proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) process, and metastasis of LUAD cells, while overexpression of GNPNAT1 revealed the opposite effects. Rescue assay showed that Snai2 knockdown reversed GNPNAT1-induced LUAD cells migration, invasion, and EMT. Mechanistically, GNPNAT1 promoted cancer cell metastasis via repressing ubiquitination degradation of Snai2 in LUAD.
CONCLUSIONS: Taken together, these data indicate that GNPNAT1 serves as a prognostic biomarker for LUAD patient. Additionally, GNPNAT1 is critical for promoting tumorigenesis and metastasis of LUAD cells and may be a potential therapeutic target for preventing LUAD metastasis.

Aim: To explore the role of miR-181a-5p in the progression of acute kidney injury (AKI) to renal interstitial fibrosis (RIF) from the perspective of DNA methylation. Materials & methods: The role of miR-181a-5p was confirmed by collecting clinical samples, injecting miR-181a-5p agomir into tail vein, and transfecting miR-181a-5p mimic in vitro. The mechanism of miR-181a-5p\'s influence on AKI induced RIF was investigated by methylation-specific PCR, bioinformatic analysis, transcriptome sequencing and so on. Results: MiR-181a-5p plays an important role in AKI induced RIF. DNMT3b-mediated miR-181a-5p promoter hypermethylation is the main reason for the downregulation of miR-181a-5p. HDAC9 and SNAI2 are direct targets of miR-181a-5p. Conclusion: Hypermethylation of miR-181a-5p promoter mediated by DNMT3b promotes AKI induced RIF by targeting HDAC9 and SNAI2.
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Pelvic organ prolapse (POP) is a group of diseases caused by extracellular matrix (ECM) degradation in pelvic supportive tissues. Cysteine and serine rich nuclear protein 1 (CSRNP1) is involved in cell proliferation and survival regulation, and reportedly facilitates collagen breakdown in human chondrocytes. The present study aimed to probe the effect of CSRNP1 on collagen metabolism in human-derived vaginal fibroblasts. High expression of CSRNP1 was found in POP patient-derived vaginal fibroblasts in comparison to normal-derived vaginal fibroblasts. Following functional experiments revealed that CSRNP1 overexpression led to proliferation inhibition, apoptosis and collagen degradation in normal vaginal fibroblasts. In line with this, silencing of CSRNP1 inhibited hydrogen peroxide (H2O2)-triggered apoptosis, ROS generation and collagen loss in normal vaginal fibroblasts. Silencing of CSRNP1 also reduced the expression of cell senescence markers p21 and γ-H2Ax (the histone H2Ax phosphorylated at Ser139), as well as curbed collagen breakdown in normal vaginal fibroblasts caused by a DNA damage agent etoposide. Transcriptomic analysis of vaginal fibroblasts showed that differentially expressed genes affected by CSRNP1 overexpression were mainly enriched in the Wnt signaling pathway. Treatment with a Wnt pathway inhibitor DKK1 blocked CSRNP1 knockdown-caused collagen deposition. Mechanistically, CSRNP1 was identified to be a target of Snail family transcriptional repressor 2 (SNAI2). Forced expression of CSRNP1 reversed the anti-apoptotic, anti-senescent and anti-collagen loss effects of SNAI2 in normal vaginal fibroblasts exposed to H2O2 or etoposide. Our study indicates that the SNAI2/CSRNP1 axis may be a key driver in POP progression, which provides a potential therapeutic strategy for POP.

BACKGROUND: Rhabdomyosarcoma (RMS) is a rare malignancy and the most common soft tissue sarcoma in children. Vasculogenic mimicry (VM) is a novel tumor microcirculation model different from traditional tumor angiogenesis, which does not rely on endothelial cells to provide sufficient blood supply for tumor growth. In recent years, VM has been confirmed to be closely associated with tumor progression. However, the ability of RMS to form VM has not yet been reported.
METHODS: Immunohistochemistry, RT-qPCR and western blot were used to test the expression level of SNAI2 and its clinical significance. The biological function in regulating vasculogenic mimicry and malignant progression of SNAI2 was examined both in vitro and in vivo. Mass spectrometry, co-immunohistochemistry, immunofluorescence staining, and ubiquitin assays were performed to explore the regulatory mechanism of SNAI2.
RESULTS: Our study indicated that SNAI2 was abnormally expressed in patients with RMS and RMS cell lines and promoted the proliferation and metastasis of RMS. Through cell tubule formation experiments, nude mice Matrigel plug experiments, and immunohistochemistry (IHC), we confirmed that RMS can form VM and that SNAI2 promotes the formation of VM. Due to SNAI2 is a transcription factor that is not easily drugged, we used Co-IP combined with mass spectrometry to screen for the SNAI2-binding protein USP7 and TRIM21. USP7 depletion inhibited RMS VM formation, proliferation and metastasis by promoting SNAI2 degradation. We further demonstrated that TRIM21 is expressed at low levels in human RMS tissues and inhibits VM in RMS cells. TRIM21 promotes SNAI2 protein degradation through ubiquitination in the RMS. The deubiquitinase USP7 and E3 ligase TRIM21 function in an antagonistic rather than competitive mode and play a key role in controlling the stability of SNAI2 to determine the VM formation and progression of RMS.
CONCLUSIONS: Our findings reveal a previously unknown mechanism by which USP7 and TRIM21 balance the level of SNAI2 ubiquitination, determining RMS vasculogenic mimicry, proliferation, and migration. This new mechanism may provide new targeted therapies to inhibit the development of RMS by restoring TRIM21 expression or inhibiting USP7 expression in RMS patients with high SNAI2 protein levels.

Actin is a protein of central importance to many cellular functions. Its localization and activity are regulated by interactions with a high number of actin-binding proteins. In a yeast two-hybrid (Y2H) screening system, snail family transcriptional repressor 2 (SNAI2 or slug) was identified as a yet unknown potential actin-binding protein. We validated this interaction using immunoprecipitation and analyzed the functional relation between slug and actin. Since both proteins have been reported to be involved in DNA double-strand break (DSB) repair, we focused on their interaction during this process after treatment with doxorubicin or UV irradiation. Confocal microscopy elicits that the overexpression of actin fused to an NLS stabilizes complexes of slug and γH2AX, an early marker of DNA damage repair.

Liver injury stimulates hepatocyte replication and hepatic stellate cell (HSC) activation, thereby driving liver regeneration. Aberrant HSC activation induces liver fibrosis. However, mechanisms underlying liver regeneration and fibrosis remain poorly understood. Here, we identify hepatic Snai1 and Snai2 as important transcriptional regulators for liver regeneration and fibrosis. Partial hepatectomy or CCl4 treatment increases occupancies of Snai1 and Snai2 on cyclin A2 and D1 promoters in the liver. Snai1 and Snai2 in turn increase promoter H3K27 acetylation and cyclin A2/D1 expressions. Hepatocyte-specific deletion of both Snai1 and Snai2, but not one alone, suppresses liver cyclin A2/D1 expression and regenerative hepatocyte proliferation after hepatectomy or CCl4 treatments but augments CCl4-stimulated HSC activation and liver fibrosis. Conversely, Snai2 overexpression in the liver enhances hepatocyte replication and suppresses liver fibrosis after CCl4 treatment. These results suggest that hepatic Snai1 and Snai2 directly promote, via histone modifications, reparative hepatocyte replication and indirectly inhibit liver fibrosis.

The aberrant expression of Forkhead box M1 (FOXM1) has been associated with the pathological processes of Parkinson\'s disease (PD), but the upstream and downstream regulators remain poorly understood. This study sought to examine the underlying mechanism of FOXM1 in dopaminergic neuron injury in PD. Bioinformatics analysis was conducted to pinpoint the differential expression of FOXM1, which was verified in the nigral tissues of rotenone-lesioned mice and dopaminergic neuron MN9D cells. Interactions among SP1, FOXM1, SNAI2, and CXCL12 were analyzed. To evaluate their effects on dopaminergic neuron injury, the lentiviral vector-mediated manipulation of FOXM1, SP1, and CXCL12 was introduced in rotenone-lesioned mice and MN9D cells. SP1, FOXM1, SNAI2, and CXCL12 abundant expression occurred in rotenone-lesioned mice and MN9D cells. Silencing of FOXM1 delayed the rotenone-induced dopaminergic neuron injury in vitro. Mechanistically, SP1 was an upstream transcription factor of FOXM1 and upregulated FOXM1 expression, leading to increased SNAI2 and CXCL12 expression. In vivo, data confirmed that SP1 promoted dopaminergic neuron injury by activating the FOXM1/SNAI2/CXCL12 axis. Our data indicate that SP1 silencing has neuroprotective effects on dopaminergic neurons, which is dependent upon the inactivated FOXM1/SNAI2/CXCL12 axis.

Colorectal cancer (CRC) is a type of intestinal cancer that causes more than 600,000 deaths every year. Overcoming the problems of metastasis requires detailed studies to reveal the potential molecular mechanisms. This study aims to reveal the molecular mechanism of CRC metastasis involving non-coding RNA regulation. The expression profile of FTH1P3 was analyzed based on the data of TCGA-COAD patient cohorts. Q-PCR analysis was performed to validate the expression of FTH1P3 in colorectal cancer cells. JASPR was used to screen transcription factors of FTH1P3. q-ChIP analysis was used to validate the target between FTH1P3 and transcription factor. Scratch assay and transwell assay were used to evaluate the migration and invasion ability of colorectal cancer cells. FTH1P3 is highly expressed in CRC patient cohort. FTH1P3 induced migration and invasion of SW480 cell through regulating epithelial-mesenchymal transition (EMT). In addition, FTH1P3 is a direct target of SNAI2. SNAI2 promotes the expression of FTH1P3. Both FTH1P3 and SNAI2 were directly targeted and repressed by miR-218-5p. Interestingly, ectopic FTH1P3 caused a decreased miR-218-5p level and an elevated nucleic SNAI2 protein expression level. Of note, only ectopic SNAI2 protein resulted in a repressed miR-218-5p and an increased FTH1P3, whereas SNAI2 3\'UTR failed to affect the expression of miR-218-5p and FTH1P3. SNAI2 transcriptionally activates FTH1P3 expression. Both SNAI2 and FTH1P3 are targets of miR-218-5p. SNAI2/FTH1P3/miR-218-5p form a positive feedback loop in the regulation of CRC metastasis.

Rhabdomyosarcoma (RMS) is a pediatric malignancy of the muscle with characteristics of cells blocked in differentiation. NOTCH1 is an oncogene that promotes self-renewal and blocks differentiation in the fusion negative-RMS sub-type. However, how NOTCH1 expression is transcriptionally maintained in tumors is unknown. Analyses of SNAI2 and CTCF chromatin binding and HiC analyses revealed a conserved SNAI2/CTCF overlapping peak downstream of the NOTCH1 locus marking a sub-topologically associating domain (TAD) boundary. Deletion of the SNAI2-CTCF peak showed that it is essential for NOTCH1 expression and viability of FN-RMS cells. Reintroducing constitutively activated NOTCH1-ΔE in cells with the SNAI2-CTCF peak deleted restored cell-viability. Ablation of SNAI2 using CRISPR/Cas9 reagents resulted in the loss of majority of RD and SMS-CTR FN-RMS cells. However, the few surviving clones that repopulate cultures have recovered NOTCH1. Cells that re-establish NOTCH1 expression after SNAI2 ablation are unable to differentiate robustly as SNAI2 shRNA knockdown cells; yet, SNAI2-ablated cells continued to be exquisitely sensitive to ionizing radiation. Thus, we have uncovered a novel mechanism by which SNAI2 and CTCF maintenance of a sub-TAD boundary promotes rather than represses NOTCH1 expression. Further, we demonstrate that SNAI2 suppression of apoptosis post-radiation is independent of SNAI2/NOTCH1 effects on self-renewal and differentiation.