The Role of Ubiquitination and the E3 Ligase Nedd4 in Regulating Corneal Epithelial Wound Healing.

Purpose: Ubiquitination serves as a fundamental post-translational modification in numerous cellular events. Yet, its role in regulating corneal epithelial wound healing (CEWH) remains elusive. This study endeavored to determine the function and mechanism of ubiquitination in CEWH. Methods: Western blot and immunoprecipitation were used to discern ubiquitination alterations during CEWH in mice. Interventions, including neuronally expressed developmentally downregulated 4 (Nedd4) siRNA and proteasome/lysosome inhibitor, assessed their impact on CEWH. In vitro analyses, such as the scratch wound assay, MTS assay, and EdU staining, were conducted to gauge cell migration and proliferation in human corneal epithelial cells (HCECs). Moreover, transfection of miR-30/200 coupled with a luciferase activity assay ascertained their regulatory mechanism on Nedd4. Results: Global ubiquitination levels were markedly increased during the mouse CEWH. Importantly, the application of either proteasomal or lysosomal inhibitors notably impeded the healing process both in vivo and in vitro. Furthermore, Nedd4 was identified as an essential E3 ligase for CEWH. Nedd4 expression was significantly upregulated during CEWH. In vivo studies revealed that downregulation of Nedd4 substantially delayed CEWH, whereas further investigations underscored its role in regulating cell proliferation and migration, through the Stat3 pathway by targeting phosphatase and tensin homolog (PTEN). Notably, our findings pinpointed miR-30/200 family members as direct regulators of Nedd4. Conclusions: Ubiquitination holds pivotal significance in orchestrating CEWH. The critical E3 ligase Nedd4, under the regulatory purview of miR-30 and miR-200, facilitates CEWH through PTEN-mediated Stat3 signaling. This revelation sheds light on a prospective therapeutic target within the realm of CEWH.

NSUN2-mediated RNA m5C modification modulates uveal melanoma cell proliferation and migration.

RNA 5-methylcytosine (m5C) is a widespread post-transcriptional modification involved in diverse biological processes through controlling RNA metabolism. However, its roles in uveal melanoma (UM) remain unknown. Here, we describe the biological roles and regulatory mechanisms of RNA m5C in UM. Initially, we identified significantly elevated global RNA m5C levels in both UM cells and tissue specimens using ELISA assay and dot blot analysis. Meanwhile, NOP2/Sun RNA methyltransferase family member 2 (NSUN2) was upregulated in both types of these samples, whereas NSUN2 knockdown significantly decreased RNA m5C level. Such declines inhibited UM cell migration and suppressed cell proliferation through cell cycle G1 arrest. Furthermore, bioinformatic analyses, m5C-RIP-qPCR, and luciferase assay identified ß-Catenin (CTNNB1) as a direct target of NSUN2-mediated m5C modification in UM cells. Additionally, overexpression of miR-124a in UM cells diminished NSUN2 expression levels indicating that it is an upstream regulator of this response. Our study suggests that NSUN2-mediated RNA m5C methylation provides a potential novel target to improve the therapeutic management of UM pathogenesis.

RNA m6 A methylation regulates uveal melanoma cell proliferation, migration, and invasion by targeting c-Met.

N6 -methyladenosine (m6 A) is a novel epitranscriptomic marker that contributes to regulating diverse biological processes through controlling messenger RNA metabolism. However, it is unknown if m6 A RNA methylation affects uveal melanoma (UM) development. To address this question, we probed its function and molecular mechanism in UM. Initially, we demonstrated that global RNA m6 A methylation levels were dramatically elevated in both UM cell lines and clinical specimens. Meanwhile, we found that METTL3, a main m6 A regulatory enzyme, was significantly increased in UM cells and specimens. Subsequently, cycloleucine (Cyc) or METTL3 targeted small interfering RNA was used to block m6 A methylation in UM cells. We found that Cyc or silencing METTL3 significantly suppressed UM cell proliferation and colony formation through cell cycle G1 arrest, as well as migration and invasion by functional analysis. On the other hand, overexpression of METTL3 had the opposite effects. Furthermore, bioinformatics and methylated RNA immunoprecipitation-quantitative polymerase chain reaction identified c-Met as a direct target of m6 A methylation in UM cells. In addition, western blot analysis showed that Cyc or knockdown of METTL3 downregulated c-Met, p-Akt, and cell cycle-related protein levels in UM cells. Taken together, our results demonstrate that METTL3-mediated m6 A RNA methylation modulates UM cell proliferation, migration, and invasion by targeting c-Met. Such a modification acts as a critical oncogenic regulator in UM development.

Paternal bisphenol a diet changes prefrontal cortex proteome and provokes behavioral dysfunction in male offspring.

Relatively little attention has been given paternal effects on next generation. Given that Bisphenol A (BPA), a ubiquitous compound in maternal diet, may disrupt brain development and behavior, we hypothesized that paternal BPA diet (PBD) could affect offspring development. Prefrontal cortex (PFC), a vital brain region, is involved in emotion and social behavior. To test whether PBD could alter developing PFC, we carried out a proteomics approach for PFC in male juvenile offspring that responded to PBD (50 mg BPA/kg diet). We found that PBD altered the expressions of binding immunoglobulin protein (BIP), CCAAT/-enhancer-binding protein homologous protein (CHOP) and B-cell lymphoma-2 (BCL-2), which could reflect endoplasmic reticulum (ER) stress. In addition, downregulation of myelinogenesis genes and myelin basic protein (MBP) could provoke myelin deficiency. Furthermore, PBD significantly increased anxiety-like behavior and impaired social behavior in male offspring. Taken together, these results revealed the alterations of ER stress and myelin destruction related molecules induced by PBD might be a potential mechanism for the behavior deficits in their male offspring. These findings remind us of the importance of paternal effects in the further environmental exposure research.

Chronic fluoride exposure-induced testicular toxicity is associated with inflammatory response in mice.

Previous studies have indicated that fluoride (F) can affect testicular toxicity in humans and rodents. However, the mechanism underlying F-induced testicular toxicity is not well understood. This study was conducted to evaluate the sperm quality, testicular histomorphology and inflammatory response in mice followed F exposure. Healthy male mice were randomly divided into four groups with sodium fluoride (NaF) at 0, 25, 50, 100 mg/L in the drinking water for 180 days. At the end of the exposure, significantly increased percentage of spermatozoa abnormality was found in mice exposed to 50 and 100 mg/L NaF. Disorganized spermatogenic cells, vacuoles in seminiferous tubules and loss and shedding of sperm cells were also observed in the NaF treated group. In addition, chronic F exposure increased testicular interleukin-17(IL-17), interleukin-17 receptor C (IL-17RC), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in transcriptional levels, as well as IL-17 and TNF-α levels in translational levels. Interestingly, we observed that F treated group elevated testicular inducible nitric oxide synthase (iNOS) mRNA level and nitric oxide (NO) concentration. Taken together, these results indicated that testicular inflammatory response could contribute to chronic F exposure induced testicular toxicity in mice.

Impairment of object recognition memory by maternal bisphenol A exposure is associated with inhibition of Akt and ERK/CREB/BDNF pathway in the male offspring hippocampus.

Bisphenol A (BPA) is a commonly used endocrine-disrupting chemical used as a component of polycarbonates plastics that has potential adverse effects on human health. Exposure to BPA during development has been implicated in memory deficits, but the mechanism of action underlying the effect is not fully understood. In this study, we investigated the effect of maternal exposure to BPA on object recognition memory and the expressions of proteins important for memory, especially focusing on the ERK/CREB/BDNF pathway. Pregnant Sprague-Dawley female rats were orally treated with either vehicle or BPA (0.05, 0.5, 5 or 50 mg/kg BW/day) during days 9-20 of gestation. Male offspring were tested on postnatal day 21 with the object recognition task. Recognition memory was assessed using the object recognition index (index=the time spent exploring the novel object/(the time spent exploring the novel object+the time spent exploring the familiar object)). In the test session performed 90 min after the training session, BPA-exposed male offspring not only spent more time in exploring the familiar object at the highest dose than the control, but also displayed a significantly decreased the object recognition index at the doses of 0.5, 5 and 50 mg/kg BW/day. During the test session performed 24h after the training session, BPA-treated males did not change the time spent exploring the familiar object, but had a decreased object recognition index at 5 and 50 mg/kg BW/day, when compared to control group. These findings indicate that object recognition memory was susceptible to maternal BPA exposure. Western blot analysis of hippocampi from BPA-treated male offspring revealed a decrease in Akt, phospho-Akt, p44/42 MAPK and phospho-p44/42 MAPK protein levels, compared to controls. In addition, BPA significantly inhibited the levels of phosphorylation of CREB and BDNF in the hippocampus. Our results show that maternal BPA exposure may full impair object recognition memory, and that impairment may be related to a decrease in Akt activation and an inhibition of the ERK/CREB/BDNF pathway in the hippocampus. This study also adds new evidence that suggests BPA has an antagonistic effect on the action of estrogen in the brain.

Maternal bisphenol a diet induces anxiety-like behavior in female juvenile with neuroimmune activation.

Maternal Bisphenol A (BPA) diet triggers anxiety in rodents, but the underlying mechanism is still unclear. Accumulating epidemiological and experimental data have demonstrated that the anxiety is associated with aberrant neuroimmune response. In this study, we found that maternal BPA diet (MBD) exacerbated anxiety-like behavior in female juvenile mice, and the molecular evidence further showed that this behavioral phenotype was connected to the neuroimmune activation, such as elevated tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 levels in prefrontal cortex (PFC) rather than in peripheral blood, which indicated that neuroimmune response might be ascribed to neuroglial activation because activated neuroglia cells could secrete proinflammatory cytokines. Subsequently, we found that ionized calcium-binding adapter molecule (Iba)-1 as a selective marker for microglia and glial fibrillary acidic protein as a specific marker for astrocyte were significantly increased at transcriptional and translational levels, which confirmed the neuroglial activation in this model. Therefore, we conclude that MBD induces excessive anxiety-like behavior in female juvenile with elevated TNF-α and IL-6 levels, as well as activated microglia and astrocyte in PFC. Herein caution must be taken to prevent potential risks from MBD becuase exacerbated anxiety-like behavior in female juvenile by MBD may be a critical contribution for subsequent growth or mental disorders.