Asparagine endopeptidase deficiency mitigates radiation-induced brain injury by suppressing microglia-mediated neuronal senescence.

Mounting evidence supports the role of neuroinflammation in radiation-induced brain injury (RIBI), a chronic disease characterized by delayed and progressive neurological impairment. Asparagine endopeptidase (AEP), also known as legumain (LGMN), participates in multiple malignancies and neurodegenerative diseases and may potentially be involved in RIBI. Here, we found AEP expression was substantially elevated in the cortex and hippocampus of wild-type (Lgmn+/+) mice following whole-brain irradiation. Lgmn knockout (Lgmn-/-) alleviated neurological impairment caused by whole-brain irradiation by suppressing neuronal senescence. Bulk RNA and metabolomic sequencing revealed AEP's involvement in the antigen processing and presentation pathway and neuroinflammation. This was further confirmed by co-culturing Lgmn+/+ primary neurons with the conditioned media derived from irradiated Lgmn+/+ or Lgmn-/- primary microglia. Furthermore, esomeprazole inhibited the enzymatic activity of AEP and RIBI. These findings identified AEP as a critical factor of neuroinflammation in RIBI, highlighting the prospect of targeting AEP as a therapeutic approach.

AEP-cleaved DDX3X induces alternative RNA splicing events to mediate cancer cell adaptation in harsh microenvironments.

Oxygen and nutrient deprivation are common features of solid tumors. Although abnormal alternative splicing (AS) has been found to be an important driving force in tumor pathogenesis and progression, the regulatory mechanisms of AS that underly the adaptation of cancer cells to harsh microenvironments remain unclear. Here, we found that hypoxia- and nutrient deprivation-induced asparagine endopeptidase (AEP) specifically cleaved DDX3X in a HIF1A-dependent manner. This cleavage yields truncated carboxyl-terminal DDX3X (tDDX3X-C), which translocates and aggregates in the nucleus. Unlike intact DDX3X, nuclear tDDX3X-C complexes with an array of splicing factors and induces AS events of many pre-mRNAs; for example, enhanced exon skipping (ES) in exon 2 of the classic tumor suppressor PRDM2 leads to a frameshift mutation of PRDM2. Intriguingly, the isoform ARRB1-Δexon 13 binds to glycolytic enzymes and regulates glycolysis. By utilizing in vitro assays, glioblastoma organoids, and animal models, we revealed that AEP/tDDX3X-C promoted tumor malignancy via these isoforms. More importantly, high AEP/tDDX3X-C/ARRB1-Δexon 13 in cancerous tissues was tightly associated with poor patient prognosis. Overall, our discovery of the effect of AEP-cleaved DDX3X switching on alternative RNA splicing events identifies a mechanism in which cancer cells adapt to oxygen and nutrient shortages and provides potential diagnostic and/or therapeutic targets.

Novel Bimolecular Fluorescence Complementation (BiFC) Assay for Visualization of the Protein-Protein Interactions and Cellular Protein Complex Localizations.

Investigations of protein-protein interactions (PPIs) are of paramount importance for comprehending cellular processes within biological systems. The bimolecular fluorescence complementation (BiFC) assay presents a convenient methodology for visualizing PPIs within live cells. While a range of fluorescent proteins have been introduced into the BiFC system, there is a growing demand for new fluorescent proteins to accommodate the expanding requirements of researchers. This study describes the introduction of Tagged blue fluorescent protein 2 (TagBFP2) into the BiFC assay to verify the interaction between two proteins, with Enhanced yellow fluorescent protein (EYFP) employed as a positive control. Both fluorescent proteins demonstrated optimal performance in this study. Compared to EYFP, the BiFC system utilizing TagBFP2 yielded a higher signal-to-noise ratio, which facilitated differentiation of the signal of PPIs from noise and enabled employment of other fluorescent proteins within the BiFC assay. Notably, the utilization of a fluorescent secondary antibody in immunofluorescence applications or the tagging of an interest protein with a fluorescent protein occupied the green or yellow channel. Overall, the present article introduces a BiFC assay that is highly straightforward, reliable, and replicable, with the ability to be completed within 1 week. This method requires neither expensive instrumentation nor technical skills of a high order.

Circular RNA hsa_circ_0075323 promotes glioblastoma cells proliferation and invasion via regulation of autophagy.

BACKGROUND: Protein p62 (sequestosome 1) encoded by gene SQSTM1 plays a vital role in mediating protectively selective autophagy in tumor cells under stressed conditions. CircSQSTM1 (hsa_circ_0075323) is a circular transcript generated from gene SQSTM1 (chr5:179260586-179260782) by back-splicing. However, the potential role of hsa_hsa_circ_0075323 in glioblastoma (GBM) remains unclear. Here, we aimed to explore the biological function of hsa_circ_0075323 in GBM and its relationship with autophagy regulation. RESULTS: Hsa_circ_0075323 is highly expressed in GBM cells and mainly locates in the cytoplasm. Inhibition of hsa_circ_0075323 in U87-MG and T98G cells attenuated proliferation and invasion ability significantly, while upregulation of has_ circ_0075323 enhanced proliferation and migration of U251-MG and A172 cells. Mechanistically, depletion of hsa_circ_0075323 in GBM cells resulted in impaired autophagy, as indicated by increased expression of p62 and decreased expression of LC3B. CONCLUSIONS: Hsa_circ_0075323 regulates p62-mediated autophagy pathway to promote GBM progression and may serve as a prognostic biomarker potentially.

Cleavage of tropomodulin-3 by asparagine endopeptidase promotes cancer malignancy by actin remodeling and SND1/RhoA signaling.

BACKGROUND: Abnormal proliferation and migration of cells are hallmarks of cancer initiation and malignancy. Asparagine endopeptidase (AEP) has specific substrate cleavage ability and plays a pro-cancer role in a variety of cancers. However, the underlying mechanism of AEP in cancer proliferation and migration still remains unclear. METHODS: Co-immunoprecipitation and following mass spectrometry were used to identify the substrate of AEP. Western blotting was applied to measure the expression of proteins. Single cell/nuclear-sequences were done to detect the heterogeneous expression of Tmod3 in tumor tissues. CCK-8 assay, flow cytometry assays, colony formation assay, Transwell assay and scratch wound-healing assay were performed as cellular functional experiments. Mouse intracranial xenograft tumors were studied in in vivo experiments. RESULTS: Here we showed that AEP cleaved a ubiquitous cytoskeleton regulatory protein, tropomodulin-3 (Tmod3) at asparagine 157 (N157) and produced two functional truncations (tTmod3-N and tTmod3-C). Truncated Tmod3 was detected in diverse tumors and was found to be associated with poor prognosis of high-grade glioma. Functional studies showed that tTmod3-N and tTmod3-C enhanced cancer cell migration and proliferation, respectively. Animal models further revealed the tumor-promoting effects of AEP truncated Tmod3 in vivo. Mechanistically, tTmod3-N was enriched in the cell cortex and competitively inhibited the pointed-end capping effect of wild-type Tmod3 on filamentous actin (F-actin), leading to actin remodeling. tTmod3-C translocated to the nucleus, where it interacted with Staphylococcal Nuclease And Tudor Domain Containing 1 (SND1), facilitating the transcription of Ras Homolog Family Member A/Cyclin Dependent Kinases (RhoA/CDKs). CONCLUSION: The newly identified AEP-Tmod3 protease signaling axis is a novel "dual-regulation" mechanism of tumor cell proliferation and migration. Our work provides new clues to the underlying mechanisms of cancer proliferation and invasive progression and evidence for targeting AEP or Tmod3 for therapy.

Circular RNA circLGMN facilitates glioblastoma progression by targeting miR-127-3p/LGMN axis.

Glioblastoma (GBM) is one of the most devastating cancers and is characterized by rapid cell proliferation and aggressive invasiveness. Legumain (LGMN), a substrate-specific protease, is associated with poor progression of GBM. Circular RNAs (circRNAs) are aberrantly expressed in various cancers and play crucial roles in tumor progression; however, the functional roles of circRNAs originating from LGMN remain largely unknown in GBM. Herein, we found that hsa_circ_0033009 (circLGMN) was the most abundantly expressed circRNA derived from LGMN. CircLGMN was upregulated in high-grade glioma (HGG), and high expression of circLGMN was associated with poor prognosis in patients with glioma. CircLGMN overexpression promoted GBM cell proliferation and enhanced cell invasion. Mechanistically, circLGMN acts as a sponge for miR-127-3p, and prevents miR-127-3p-mediated degradation of LGMN mRNA, ultimately leading to increased LGMN protein expression. Treatment with miR-127-3p mimic suppressed proliferation and reduced invasion of GBM cells overexpressing circLGMN. Moreover, circLGMN overexpression promoted GBM malignancy in vivo, while miR-127-3p overexpression alleviated this effect. Taken together, circLGMN is a novel tumor-promoting circRNA that acts by sponging miR-127-3p, which ultimately leads to LGMN upregulation. Thus, targeting the circLGMN/miR-127-3p/LGMN axis might be a promising strategy for GBM treatment. More importantly, the discovery of the self-regulatory mechanism of LGMN expression by circLGMN, will facilitate further research on LGMN.

Preliminary study on the feasibility of a two-stage screening strategy for otitis media with effusion in children.

AIM: To evaluate the feasibility of a two-stage screening strategy for otitis media with effusion (OME) in pre-school and school children. The risk factors of OME were also studied. METHODS: One hundred and eighty-nine children aged 4-8 years were recruited. The two-stage screening consisted of an on-site screening with a portable otoscopy along with a questionnaire to both diagnose children with OME and identify children at risk, and a standard screening performed at a regional hospital for final diagnosis. The prevalence detected from the two-stage screening approach was compared to the actual prevalence. RESULTS: The detection rate of OME through the two-stage screening approach was not significantly different from the actual prevalence rate (12.7% vs. 13.4%, P = 0.847). Children from the urban area had a lower risk for OME than that from the rural area (P = 0.007, odds ratio (OR) = 0.28, 95% confidence interval (CI): 0.11-0.74). Compared to childcare dining, family dining helped to reduce the chance of OME (P < 0.001, OR = 0.15, 95% CI: 0.06-0.38). CONCLUSIONS: The two-stage screening strategy was effective for screening for OME among pre-school and school children. It can be used in rural areas that have a high prevalence of OME and limited medical resources.

Liver X receptor activation reduces gastric cancer cell proliferation by suppressing Wnt signalling via LXRß relocalization.

Liver X receptors (LXRs) are involved in various diseases associated with lipid disorders, and in regulating cancer cell proliferation. However, the underlying molecular mechanisms, especially those in gastric cancer (GC) remain to be clarified. In this study, immunohistochemistry analysis revealed that LXRß was mainly expressed in GC tissue, with less expression in adjacent normal tissues. The LXRß agonist T0901317 efficiently suppressed the proliferation and colony formation of various GC cell lines. We further showed that LXRß translocated from the cytoplasm to the nucleus when activated by T0901317. LXRß nuclear localization suppressed the activation of Wnt signalling and decreased the expression of target genes such as MYC, BMP4, and MMP7 through binding to their promoters. Moreover, we demonstrated that the LXR agonist efficiently suppressed GC tumour growth in a nude mouse xenograft model. Taken together, these results revealed that LXRß agonist inhibited GC cells proliferation by suppressing Wnt signalling via LXRß relocalization. The results strongly suggest that LXRß could be a promising target in GC therapy.

Green Tea Polyphenols Protects Cochlear Hair Cells from Ototoxicity by Inhibiting Notch Signalling.

Notch signalling pathway plays an essential role in the development of cochlea, which inhibits the proliferation of hair cells. Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea, which presents strong antioxidant activation and has been applied for anti-cancer and anti-inflammatory. In this study, we treated the cochlear explant cultures with EGCG and found that EGCG can protect cochlear hair cells from ototoxic drug gentamicin. We demonstrated that EGCG could down-regulate the expression of Notch signalling pathway target genes, such as Hes1, Hes5, Hey1 and Hey5. However, the Notch pathway ligands such as Delta1, Jag1 and Jag2 were not affected by EGCG. To further illustrate the mechanism of recover cochlear hair cells, we demonstrated that EGCG inhibited the activity of γ-secrectase to suppress Notch signalling pathway and promoted the proliferation and regeneration of hair cells in the damaged cochlea. Our results suggest for the first time the role of EGCG as an inhibitor of the Notch signalling pathway, and support its potential value in hearing-impaired recovery in clinical therapy.