UBE2M, also known as UBC12, is one of two neddylation conjugation enzymes. The in vitro cell culture work showed that UBE2M is essential for the growth and survival of cancer cells, but its in vivo role in tumorigenesis, particularly in the tumor microenvironment (TME), remains elusive. Here, we report that in a well-established KrasG12D lung tumor mouse model, Ube2m deletion caused an inflammatory TME, severe lung edema, and early death. Mechanistically, while KrasG12D activates MAPK and mTORC1 signals for proliferation, Ube2m deletion increases mitochondrial MOMP/mPTP and inhibits Parkin neddylation and activity, leading to compromised mitophagy. The combination of KrasG12D activation and Ube2m deletion caused cytoplasmic release of damaged mitochondrial DNA to activate the cGAS-STING-NF-κB pathway, followed by inflammatory cytokine secretion, immune cell infiltration, and inflammatory TME. This process is largely blocked by the STING antagonist H-151, indicating a causal role of the cGAS-STING axis. In human lung cancer with KRAS mutations, low UBE2M expression is correlated with high infiltrations of immune cells, demonstrating its relevance to human disease. Our study unexpectedly showed a paracrine role of Ube2m in the prevention of TME upon KrasG12D activation in the lung.

G protein-coupled receptors (GPCRs) mediate most cellular responses to hormones, neurotransmitters, and environmental stimulants. However, whether GPCRs participate in tissue homeostasis through ferroptosis remains unclear. Here we identify that GPR56/ADGRG1 renders cells resistant to ferroptosis and deficiency of GPR56 exacerbates ferroptosis-mediated liver injury induced by doxorubicin (DOX) or ischemia-reperfusion (IR). Mechanistically, GPR56 decreases the abundance of phospholipids containing free polyunsaturated fatty acids (PUFAs) by promoting endocytosis-lysosomal degradation of CD36. By screening a panel of steroid hormones, we identified that 17α-hydroxypregnenolone (17-OH PREG) acts as an agonist of GPR56 to antagonize ferroptosis and efficiently attenuates liver injury before or after insult. Moreover, disease-associated GPR56 mutants were unresponsive to 17-OH PREG activation and insufficient to defend against ferroptosis. Together, our findings uncover that 17-OH PREG-GPR56 axis-mediated signal transduction works as a new anti-ferroptotic pathway to maintain liver homeostasis, providing novel insights into the potential therapy for liver injury.

Recent advances in next-generation sequencing have revealed that long non-coding RNAs (lncRNAs) can encode functional micropeptides through small open reading frames (sORFs), altering the perception of the non-coding genome. In this study, we identified a 48-amino acid micropeptide named PAMP (proline-associated micropeptide), encoded by the lncRNA PSMA3-AS1, as a novel tumor suppressor in lung adenocarcinoma (LUAD). PAMP is significantly downregulated in LUAD tissues and positively correlates with favorable prognosis. Functional assays demonstrated that PAMP inhibits LUAD cell proliferation in vitro and suppresses tumor growth in vivo. Mechanistically, PAMP directly interacts with PYCR1, a key enzyme in proline biosynthesis. Structural modeling and mutagenesis revealed that the PAMP-F16 and PYCR1-N123 residues are critical for the interaction, resulting in the inhibition of PYCR1 enzymatic activity and decreased proline accumulation. Notably, synthetic PAMP administration recapitulates these anti-tumor effects, effectively reducing intracellular proline levels and impairing tumor progression in cellular and animal models. Together, our findings uncover a previously uncharacterized lncRNA-encoded micropeptide that orchestrates proline metabolic reprogramming to restrain LUAD development, offering new opportunities for metabolic intervention in precision oncology.

Elevated temperatures are a major environmental stress factor that impairs rice productivity and compromises grain quality. This study investigated the effects of natural allelic variation in soluble starch synthase I (SSI) on grain quality and starch structural properties under high-temperature (HT) conditions. Four near-isogenic lines (NILs) differing in SSI alleles and carrying either the Wxb or wx backgrounds were cultivated under HT stress. A range of analytical techniques revealed that HT significantly reduced grain appearance quality, protein content, apparent amylose content (AAC), and total starch content across all NILs. In addition, rapid visco-analysis (RVA) profiles of rice flour showed a marked reduction in viscosity under HT. Starch fine structure analysis demonstrated a decrease in short-chain amylopectin and an increase in long-chain amylopectin under HT, which was associated with enhanced starch crystallinity and elevated gelatinization temperatures. Notably, rice lines carrying the SSIi allele, in both Wxb and wx backgrounds, exhibited milder declines in grain quality traits compared to those with the SSIj allele. These findings provide new insights into the role of SSI allelic variation in maintaining rice grain quality under HT stress and offer a genetic basis for breeding heat-tolerant rice varieties.

Lung cancer remains the leading cause of cancer-related death worldwide, and long noncoding RNAs (lncRNAs) have been implicated in its tumourigenesis and progression. However, the roles lncRNAs play in lung cancer remain unclear. In this study, we discovered an important role for the lncRNA MNX1 Antisense RNA 1 (MNX1-AS1) as a critical regulator of one-carbon metabolism reprogramming. Glutamine depletion altered chromatin accessibility, leading to downregulation of MNX1-AS1, while elevated expression of MNX1-AS1 was correlated with poor prognosis in patients with non-small cell lung cancer. Functional studies showed that MNX1-AS1 promoted cell proliferation and sphere formation in vitro, and subcutaneous and orthotopic tumour growth in vivo. Mechanistically, MNX1-AS1 directly binds to calcyclin binding protein (CACYBP), protecting it from ubiquitin-mediated degradation; thus, the MNX1-AS1/CACYBP complex accelerates the transcription of key one-carbon metabolism-related genes through the Wnt/β-catenin pathway. MNX1-AS1/CACYBP/β-catenin axis upregulated key one-carbon metabolism-related genes, which were essential for generating related metabolites and maintaining cellular redox balance to support lung cancer cell proliferation. These findings established that the lncRNA MNX1-AS1 acts as a crucial driver of one-carbon metabolism reprogramming in non-small cell lung cancer and highlight that the newly identified MNX1-AS1/CACYBP/β-catenin axis may serve as a potential prognostic biomarker and therapeutic target for lung cancer intervention.

tRNA-derived fragments (tRFs) are frequently dysregulated in cancers, and approaches for the detection of tRFs within biological samples are vital for their expression analysis and functional exploration. Here, we present a protocol for detecting tRFs using a modified TaqMan quantitative real-time PCR (qRT-PCR)-based technique, Dumbbell-PCR (Db-PCR). We describe steps for primer and adapter design, adapter-RNA ligation, and RNA detection. This protocol streamlines and enhances the precision of tRF quantification in cells, tissues, and plasma, facilitating a time-efficient and reliable assessment of their presence. For complete details on the use and execution of this protocol, please refer to Yu et al.1 and Sun et al.2.

Lung adenocarcinoma (LUAD) progression involves alterations in oncogenes and tumor suppressor genes, collectively shaping tumorigenic landscape. However, the precise interactions within this landscape remain inadequately understood. Here, we present a functional characterization of a novel long non-coding RNA (lncRNA), SPAT (splice associated transcript). SPAT is downregulated in LUAD and its expression positively correlates with favorable prognosis. In vitro and in vivo experiments demonstrated that SPAT inhibits the migration of LUAD cells. This inhibitory effect is mediated by SPAT’s interaction with splicing factor 1 (SF1), which disrupts SF1-mediated splicing of KITLG/SCF exon 6, thereby suppressing ERK phosphorylation. Our findings suggest that SPAT acts as a tumor suppressor in LUAD by regulating alternative splicing and highlight its potential as a therapeutic target for managing LUAD metastasis.

Ovarian cancer is an aggressive gynecological tumor usually diagnosed with widespread metastases. Extracellular vesicles (EVs), though recognized as important mediators of tumor metastasis, have received limited attention into their specific functions via the mRNA profiling. Here it is reported elevated expression and selective enrichment of INAVA mRNA in both plasma- and tissue-derived EVs from ovarian cancer patients, which is positively correlated with distant metastasis and poor prognosis. Functionally, INAVA mRNA, upon uptake and translation, activates normal ovarian fibroblasts (NOFs) and drives extensive peritoneum metastasis in the orthotopic xenograft mouse model. Mechanistically, INAVA competitively binds with high mobility group protein A2 (HMGA2) and consequently inhibit its interaction with vaccinia-related kinase 1 (VRK1), leading to reduced HMGA2 phosphorylation on Ser105. Interestingly, this inhibitory phosphorylation stabilizes HMGA2 via blocking tripartite motif-containing 21 (TRIM21) -mediated K48-linked ubiquitylation, and ultimately enhances the transcription of STAT3 to activate NOFs. Lastly, a cell-permeable peptide that disrupts the INAVA-HMGA2 interaction leads to attenuated NOF activation and provides a promising strategy for ovarian cancer therapy.

Abstract Circular RNAs (circRNAs) are a distinctive class of non‐coding RNAs with covalent closed‐loop structure, lacking 5′ caps and 3′ poly(A) tails. These molecules are prevalent in eukaryotes and play key roles in cancer. Here, the function of a new circRNA, circMETTL6, in ovarian cancer is identified and investigated. The prognostic significance of circMETTL6 is assessed using RNA in situ hybridization. Functional studies involving circMETTL6 overexpression are performed both in vitro and in vivo. Mechanistic investigations are performed using RNA‐seq, RNA pull‐down, RNA immunoprecipitation, co‐immunoprecipitation, chromatin immunoprecipitation, protein degradation assay and dual‐luciferase reporter assays. circMETTL6 is significantly downregulated in ovarian cancer, and its lower expression correlates with worse prognosis. Overexpression of circMETTL6 significantly inhibited proliferation, migration, and invasion of ovarian cancer cell in vitro, as well as tumor growth and metastasis in vivo. Mechanistically, circMETTL6 recruited the non‐POU domain containing octamer binding protein (NONO) by binding to its Coiled‐coil domain and disrupted its binding with RNA polymerase II subunit A (POLR2A), and consequently inhibiting growth differentiation factor 15 (GDF15) transcription, thereby suppressing ovarian cancer progression. These findings establish circMETTL6 as a novel tumor suppressor in ovarian cancer. Targeting the circMETTL6/NONO/GDF15 axis presents a potential therapeutic avenue for ovarian cancer treatment.

RNA modifications, particularly N6-methyladenosine (m6A), play crucial roles in gene expression regulation. While extensively studied in the context of mRNA, the impact of m6A on long non-coding RNAs (lncRNAs) remains elusive. This research aimed to reveal the regulatory landscape of m6A in lncRNA expression. In a comprehensive analysis across 6219 samples spanning 12 cancer types, we unveiled METTL3 as the most potent regulator of lncRNA expression among the examined 19 m6A regulators. A total of 397 METTL3-mediated m6A-modified lncRNAs (mmlncRs) were unveiled across 12 cancer types, indicating a consistent mechanism of METTL3-mediated lncRNA regulation. Functional assays demonstrated that METTL3 knockout significantly impeded lung cancer cell proliferation and progression. Leveraging RNA-seq and MeRIP-seq, we identified C1RL-AS1 as a bona fide m6A target of METTL3 in lung cancer, revealing its oncogenic role. Mechanistically, METTL3 depletion disrupts m6A modification on C1RL-AS1, leading to its downregulated expression. YTHDF2 binds to C1RL-AS1, maintaining its stability in a m6A-dependent manner. This study provides a valuable resource for the exploration of mmlncRs as promising therapeutic targets in cancers, shedding light on the intricate regulatory networks orchestrated by METTL3.

F-box and WD repeat domain-containing 7 (FBXW7) is a tumor suppressor that targets various oncoproteins for degradation, but its role in modulating cancer-associated fibroblasts (CAFs) in the tumor microenvironment remains elusive. Here, we report that FBXW7 expression is gradually downregulated in CAFs during the progression of human pancreatic and lung cancers. Mechanically, FBXW7 inhibits histone lysine methyltransferase 2 (KMT2) methyltransferase activity via retinoblastoma binding protein 5 (RbBP5) binding, whereas FBXW7 depletion abrogates the binding to activate KMT2, leading to increased H3K4 methylations and global upregulation of gene expression. Activation of the interleukin-17 (IL-17) signaling pathway triggers the secretion of cytokines and chemokines to promote migration, invasion, and sphere formation of lung cancer cells. Coinjection of Fbxw7-depleted mouse embryonic fibroblasts with cancer cells enhances in vivo tumor growth, demonstrating a paracrine effect. Hypoxia downregulates CAF FBXW7 via ETS proto-oncogene 1 (ETS1) to increase H3K4 methylation, whereas conditioned media from hypoxia-exposed CAFs promotes migration and invasion of pancreatic cancer cells, highlighting FBXW7's tumor-suppressing role through KMT2 inactivation.

Background To assess the status of NEUROG1 methylation in the advanced adenoma and colorectal cancer. Methods The NEUROG1 methylation in tissue and stool samples from patients with colorectal cancer (CRC), advanced adenoma (AA), and non-advanced adenoma (NAA) were evaluated using methylation-specific quantitative polymerase chain reaction (PCR). Results In tissue samples, the NEUROG1 methylation detection rates were 36% for CRC, 24% for NAA, and 88% for AA. In stool samples, the NEUROG1 methylation detection had a sensitivity of 63.46% for CRC with a positive predictive value (PPV) of 85.94%. The overall diagnostic specificity of the test for the NAA and the healthy control was 76.32%, with a negative predictive value (NPV) of 40.28%. Conclusion NEUROG1 methylation detection can potentially be used in the CRC and AA screening.

Allosteric modulation of receptor responses to endogenous agonists has therapeutic value, maintaining ligand profiles, reducing side effects and restoring mutant responses. Adhesion G-protein-coupled receptors (aGPCRs), with large N termini, are ideal for allosteric modulator development. We designed a nanobody strategy targeting ADGRG2 N-terminal fragments and got a specific nanobody Nb23-bi, which promoted dehydroepiandrosterone (DHEA)-induced ADGRG2 activation and reversed mutant-induced dysfunctions. By combining structural characterization, crosslinking mass spectrometry, mutational analysis and molecular dynamics simulations, we clarified the allosteric mechanism of how the Nb23-bi modulates conformational changes in the DHEA-binding pocket. Animal studies showed that Nb23-bi promoted the response of DHEA in alleviating testicular inflammation and reversing mutant defects. In summary, we developed an allosteric nanobody of ADGRG2 and gained insights into its functions in reversing disease-associated dysfunctions. Our study may serve as a template for developing allosteric modulators of other aGPCRs for biological and therapeutic purposes.

Persistent infection with high-risk human papillomavirus (HPV) is the primary contributor to the development of cervical cancer. Although HPV oncoproteins E6 and E7 clearly trigger cervical tumorigenesis by inactivating p53 and Rb pathways, the downstream mediators of p53/Rb inactivation remain elusive. Here we report that CDT2, a subunit of Cullin-RING ligase 4 (CRL4), is significantly upregulated in cervical carcinoma tissues, which correlates with E6/E7 expression and poor patient survival. Mechanistically, E7-mediated Rb degradation upregulates E2F1, which in turn increases CDT2 transcription, whereas E6-mediated p53 degradation downregulates TRIM22, a novel E3 ligase for CDT2 degradation, leading to CDT2 accumulation to promote growth and survival of cervical cancer cells. Importantly, CDT2 depletion induces DNA aneuploidy and senescence via stabilization of histone lysine methyltransferase SET8, a CRL4CDT2 substrate, acting as a tumor suppressor. Collectively, the TRIM22-CDT2-SET8 axis is the key mediator of the p53/Rb signals in regulation of growth and survival of HPV-positive cervical carcinoma cells, Thus, CDT2 could serve as a prognostic biomarker and therapeutic target for these carcinomas.

Equilibrioception (sensing of balance) is essential for mammals to perceive and navigate the three-dimensional world. A rapid mechanoelectrical transduction (MET) response in vestibular hair cells is crucial for detecting position and motion. Here, we identify the G protein-coupled receptor (GPCR) LPHN2/ADGRL2, expressed on the apical membrane of utricular hair cells, as essential for maintaining normal balance. Loss of LPHN2 specifically in hair cells impaired both balance behavior and the MET response in mice. Functional analyses using hair-cell-specific Lphn2-knockout mice and an LPHN2-specific inhibitor suggest that LPHN2 regulates tip-link-independent MET currents at the apical surface of utricular hair cells. Mechanistic studies in a heterologous system show that LPHN2 converts force stimuli into increased open probability of transmembrane channel-like protein 1 (TMC1). LPHN2-mediated force sensation triggers glutamate release and calcium signaling in utricular hair cells. Importantly, reintroducing LPHN2 into the hair cells of Lphn2-deficient mice restores vestibular function and MET response. Our data reveal that a mechanosensitive GPCR is required for equilibrioception.

Background:Immune checkpoint inhibitors play an important role in the treatment of solid tumors, but the currently used immune checkpoint inhibitors targeting programmed cell death-1 (PD-1), programmed cell death ligand-1 (PD-L1), and cytotoxic T-lymphocyte antigen-4 (CTLA-4) show limited clinical efficacy in many breast cancers. B7H3 has been widely reported as an immunosuppressive molecule, but its immunological function in breast cancer patients remains unclear. Methods:We analyzed the expression of B7H3 in breast cancer samples using data from the Cancer Genome Atlas Program (TCGA) and the Gene Expression Omnibus (GEO) databases. MicroRNAs were selected using the TarBase, miRTarBase, and miRBase databases. The regulatory role of the microRNA hsa-miR-214-3p on B7H3 was investigated through dual-luciferase reporter assays, which identified the specific action sites of interaction. The expression levels of B7H3 and hsa-miR-214-3p in human breast cancer tissues and adjacent normal tissues were quantified using Western blotting and quantitative PCR (qPCR). In vitro experiments were performed to observe the effects of modulating the expression of B7H3 or hsa-miR-214-3p on breast cancer cell proliferation and apoptosis. Additionally, the regulatory impact of hsa-miR-214-3p on B7H3 was examined. Enzyme-linked immunosorbent assays (ELISA) and flow cytometry were employed to assess the effects of co-cultured breast cancer cells and normal human peripheral blood mononuclear cells (PBMCs) on immune cells and associated cytokines. Results:In breast cancer tissues, the expression level of B7H3 is inversely correlated with that of hsa-miR-214-3p, as well as with the regulatory effects on breast cancercell behavior. Hsa-miR-214-3p was found to inhibit breast cancer cell growth by downregulating B7H3. Importantly, our research identified, for the first time, two binding sites for hsa-miR-214-3p on the 3' UTR of B7H3, both of which exert similar effects independently. Co-culture experiments revealed that hsa-miR-214-3p obstructs the suppressive function of B7H3 on CD8+ T cells and natural killer cells. Conclusions:This study confirms the existence of two hsa-miR-214-3p binding sites on the 3' UTR of B7H3, reinforcing the role of hsa-miR-214-3p as a regulatory factor for B7H3. In breast cancer, hsa-miR-214-3p reduces tumor cell proliferation and enhances the tumor immune microenvironment by downregulating B7H3. These findings suggest new potential targets for the clinical treatment of breast cancer.

Doublets in single-cell sequencing data, caused by the simultaneous capture of two or more cells within a single reaction volume, introduce biases that compromise downstream analysis. Existing doublet detection methods primarily focus on single-modality data and exhibit limited robustness across datasets. To overcome these limitations, we developed OmniDoublet, a multimodal doublet detection method that integrates transcriptomic and epigenomic data. OmniDoublet leverages the Jaccard similarity coefficient to calculate weights that assess the reliability of neighboring cells across modalities, combining doublet scores from different modalities into a final integrated score. It further employs a Gaussian mixture model (GMM) to establish thresholds, enabling accurate binary classification of cells as singlets or doublets based on the integrated score. OmniDoublet offers a robust framework for detecting doublets across diverse scenarios. Benchmarking against state-of-the-art methods across various datasets demonstrates that OmniDoublet achieves superior accuracy, robustness, and scalability. By harnessing the comprehensive information from multimodal single-cell data, OmniDoublet enhances doublet detection, enabling researchers to gain more accurate and reliable insights into cellular processes.

Amylose content (AC) is a crucial determinant of the eating and cooking quality (ECQ) of rice, with low AC varieties exhibiting a softer texture and greater stickiness −attributes that enhance palatability and are desirable in specific culinary contexts. To harness these traits, significant efforts have been made to manipulate AC to improve rice ECQ. Our research utilized the MutMap+ approach to identify LAC6/TL1, a gene that is an allele of Du13, responsible for low AC. LAC6 encodes a C2H2 zinc finger protein, which specifically increases the splicing efficiency of the Wxb allele without affecting the Wxa allele. Functional studies of LAC6 revealed that its proper integration could rectify the undesirable AC phenotype, whereas mutations within this gene led to reduced AC and were associated with shorter grain length and decreased thousand-grain weight. Despite these drawbacks, such mutations positively impact rice palatability, presenting a trade-off between grain size and eating quality. To address the challenges posed by the reduced grain weight associated with LAC6 mutations, we developed a specific molecular marker for LAC6, which has been effectively used in breeding programs to select lac6/tl1/du13 homozygous individuals with larger grain size. Our findings demonstrate that the “small grain” trait associated with lac6/tl1/du13 can be effectively mitigated through combined phenotype-based and marker-assisted selection. This study highlights the potential of lac6/tl1/du13 as a valuable gene for breeding novel, high-quality soft rice varieties through targeted breeding strategies.

Objective. Ovarian cancer is the deadliest gynecologic malignancy worldwide. Ultrasound is the most useful non-invasive test for preoperative diagnosis of ovarian cancer. In this study, by leveraging multiple ultrasound images from the same patient to generate personalized, informative statistical radiomic features, we aimed to develop improved ultrasound image-based prognostic models for ovarian cancer.Approach. A total of 2057 ultrasound images from 514 ovarian cancer patients, including 355 patients with epithelial ovarian cancer, from two hospitals in China were collected for this study. The models were constructed using our recently developed Frequency Appearance in Multiple Univariate pre-Screening feature selection algorithm and Cox proportional hazards model.Main results. The models showed high predictive performance for overall survival (OS) and recurrence-free survival (RFS) in both epithelial and nonepithelial ovarian cancer, with concordance indices ranging from 0.773 to 0.794. Radiomic scores predicted 2 year OS and RFS risk groups with significant survival differences (log-rank test,P< 1.0 × 10-4for both validation cohorts). OS and RFS hazard ratios between low- and high-risk groups were 15.994 and 30.692 (internal cohort) and 19.339 and 19.760 (external cohort), respectively. The improved performance of these newly developed prognostic models was mainly attributed to the use of multiple preoperative ultrasound images from the same patient to generate statistical radiomic features, rather than simply using the largest tumor region of interest among them. The models also revealed that the roundness of tumor lesion shape was positively correlated with prognosis for ovarian cancer.Significance.The newly developed prognostic models based on statistical radiomic features from ultrasound images were highly predictive of the risk of cancer-related death and possible recurrence not only for patients with epithelial ovarian cancer but also for those with nonepithelial ovarian cancer. They thereby provide reliable, non-invasive markers for individualized prognosis evaluation and clinical decision-making for patients with ovarian cancer.