Emerging roles of RNA-binding proteins in fatty liver disease.

A rampant and urgent global health issue of the 21st century is the emergence and progression of fatty liver disease (FLD), including alcoholic fatty liver disease and the more heterogenous metabolism-associated (or non-alcoholic) fatty liver disease (MAFLD/NAFLD) phenotypes. These conditions manifest as disease spectra, progressing from benign hepatic steatosis to symptomatic steatohepatitis, cirrhosis, and, ultimately, hepatocellular carcinoma. With numerous intricately regulated molecular pathways implicated in its pathophysiology, recent data have emphasized the critical roles of RNA-binding proteins (RBPs) in the onset and development of FLD. They regulate gene transcription and post-transcriptional processes, including pre-mRNA splicing, capping, and polyadenylation, as well as mature mRNA transport, stability, and translation. RBP dysfunction at every point along the mRNA life cycle has been associated with altered lipid metabolism and cellular stress response, resulting in hepatic inflammation and fibrosis. Here, we discuss the current understanding of the role of RBPs in the post-transcriptional processes associated with FLD and highlight the possible and emerging therapeutic strategies leveraging RBP function for FLD treatment. This article is categorized under: RNA in Disease and Development > RNA in Disease.

YTHDF2 promotes gastric cancer progression and enhances chemoradiotherapy resistance.

The role of YTHDF2 in gastric cancer (GC) is controversial. Due to the limitations of technical difficulty and experimental period, research on completely knocking out YTHDF2 is rare. Therefore, further investigations are still needed to clarify the YTHDF2's clinical significance and biological function in GC. To carry out the investigation, an analysis was performed on the expression levels of YTHDF2 in both publicly available databases and samples obtained from patients with gastric cancer. Based on the complete knockout of YTHDF2 using the CRISPR-Cas9 system, in vivo and in vitro experiments were conducted to analyze the effects of YTHDF2 on tumor formation, radiotherapy and chemoradiotherapy resistance in GC. Our investigation revealed an increase in YTHDF2 levels in GC tissues, which was found to be associated with a negative prognosis. Under hypoxic conditions, high expression of YTHDF2 enhanced the invasion of gastric cancer cells, and high expression of YTHDF2 was associated with HIF-1a. YTHDF2 facilitated gastric cancer cell growth in vitro and in vivo. Moreover, the results of the present study demonstrated that YTHDF2 mediated the expression of CyclinD1 and stability of CyclinD1 mRNA. CyclinD1 knockdown inhibited YTHDF2-mediated GC cell proliferation whereas CyclinD1 overexpression ameliorated YTHDF2 knockdown-induced inhibition of GC progression. Furthermore, YTHDF2 also promoted resistance to DDP and CTX chemotherapy, along with radiotherapy treatment for GC cells. The findings suggested that YTHDF2 expression accelerated GC progression through a potential mechanism involving CyclinD1 expression, and enhanced chemoradiotherapy resistance. This indicated that YTHDF2 could be a promising prognostic biomarker and therapeutic target for individuals diagnosed with GC.

Understanding YTHDF2-mediated mRNA degradation by m6A-BERT-Deg.

N6-methyladenosine (m6A) is the most abundant mRNA modification within mammalian cells, holding pivotal significance in the regulation of mRNA stability, translation and splicing. Furthermore, it plays a critical role in the regulation of RNA degradation by primarily recruiting the YTHDF2 reader protein. However, the selective regulation of mRNA decay of the m6A-methylated mRNA through YTHDF2 binding is poorly understood. To improve our understanding, we developed m6A-BERT-Deg, a BERT model adapted for predicting YTHDF2-mediated degradation of m6A-methylated mRNAs. We meticulously assembled a high-quality training dataset by integrating multiple data sources for the HeLa cell line. To overcome the limitation of small training samples, we employed a pre-training-fine-tuning strategy by first performing a self-supervised pre-training of the model on 427 760 unlabeled m6A site sequences. The test results demonstrated the importance of this pre-training strategy in enabling m6A-BERT-Deg to outperform other benchmark models. We further conducted a comprehensive model interpretation and revealed a surprising finding that the presence of co-factors in proximity to m6A sites may disrupt YTHDF2-mediated mRNA degradation, subsequently enhancing mRNA stability. We also extended our analyses to the HEK293 cell line, shedding light on the context-dependent YTHDF2-mediated mRNA degradation.

Clinical phenotype of a Kallmann syndrome patient with IL17RD and CPEB4 variants.

Background: This study aimed to characterize the clinical phenotype and genetic variations in patients with Kallmann syndrome (KS). Methods: This study involved the collection and analysis of clinical data from an individual with sporadic KS. Following this, peripheral blood samples were obtained from the patient and his parents. Genomic deoxyribonucleic acid was extracted and subjected to whole-exome sequencing and genomic copy number variation (CNV) detection. Finally, Sanger sequencing was performed to validate the suspected pathogenic variants. Results: Whole-exome sequencing confirmed that the child carried both the IL17RD variant (c.2101G>A, p.Gly701Ser) inherited from the mother and the new CPEB4 variant (c.1414C>T, p.Arg472*). No pathogenic CNVs were identified in CNV testing. Conclusion: Bioinformatics analysis shows that the IL17RD protein undergoing Gly701Ser mutation and is speculated to be phosphorylated and modified, thereby disrupting fibroblast growth factor signaling. This study also suggested that the CPEB4 might play a crucial role in the key signaling process affecting olfactory bulb morphogenesis. Overall, the findings of this study broaden the gene expression profile of KS-related pathogenic genes. This offers a new avenue for exploring the pathogenic mechanism of KS and provides valuable insights for precise clinical diagnosis and treatment strategies for this condition.

m6A-dependent upregulation of DDX21 by super-enhancer-driven IGF2BP2 and IGF2BP3 facilitates progression of acute myeloid leukaemia.

BACKGROUND: Acute myeloid leukaemia (AML) is a haematological malignancy with unfavourable prognosis. Despite the effectiveness of chemotherapy and targeted therapy, relapse or drug resistance remains a major threat to AML patients. N6-methyladenosine (m6A) RNA methylation and super-enhancers (SEs) are extensively involved in the leukaemogenesis of AML. However, the potential relationship between m6A and SEs in AML has not been elaborated. METHODS: Chromatin immunoprecipitation (ChIP) sequencing data from Gene Expression Omnibus (GEO) cohort were analysed to search SE-related genes. The mechanisms of m6 A-binding proteins IGF2BP2 and IGF2BP3 on DDX21 were explored via methylated RNA immunoprecipitation (MeRIP) assays, RNA immunoprecipitation (RIP) assays and luciferase reporter assays. Then we elucidated the roles of DDX21 in AML through functional assays in vitro and in vivo. Finally, co-immunoprecipitation (Co-IP) assays, RNA sequencing and ChIP assays were performed to investigate the downstream mechanisms of DDX21. RESULTS: We identified two SE-associated transcripts IGF2BP2 and IGF2BP3 in AML. High enrichment of H3K27ac, H3K4me1 and BRD4 was observed in IGF2BP2 and IGF2BP3, whose expression were driven by SE machinery. Then IGF2BP2 and IGF2BP3 enhanced the stability of DDX21 mRNA in an m6A-dependent manner. DDX21 was highly expressed in AML patients, which indicated a poor survival. Functionally, knockdown of DDX21 inhibited cell proliferation, promoted cell apoptosis and led to cell cycle arrest. Mechanistically, DDX21 recruited transcription factor YBX1 to cooperatively trigger ULK1 expression. Moreover, silencing of ULK1 could reverse the promoting effects of DDX21 overexpression in AML cells. CONCLUSIONS: Dysregulation of SE-IGF2BP2/IGF2BP3-DDX21 axis facilitated the progression of AML. Our findings provide new insights into the link between SEs and m6A modification, elucidate the regulatory mechanisms of IGF2BP2 and IGF2BP3 on DDX21, and reveal the underlying roles of DDX21 in AML.

Mex-3 RNA binding family member A (MEX3A)/circMPP6 complex promotes colorectal cancer progression by inhibiting autophagy.

RNA-binding proteins (RBPs)-RNA networks have contributed to cancer development. Circular RNAs (circRNAs) are considered as protein recruiters; nevertheless, the patterns of circRNA-protein interactions in colorectal cancer (CRC) are still lacking. Processing bodies (PBs) formed through liquid-liquid phase separation (LLPS) are membrane-less organelles (MLOs) consisting of RBPs and RNA. Previous evidence suggests a connection between PBs dynamics and cancer progression. Despite the increasingly acknowledged crucial role of RBPs and RNA in the accumulation and maintenance of MLOs, there remains a lack of specific research on the interactions between PBs-related RBPs and circRNAs in CRC. Herein, we identify that MEX-3 RNA binding family member A (MEX3A), frequently upregulated in CRC tissues, predicts poorer patient survival. Elevated MEX3A accelerates malignance and inhibits autophagy of CRC cells. Importantly, MEX3A undergoes intrinsically disordered regions (IDRs)-dependent LLPS in the cytoplasm. Specifically, circMPP6 acts as a scaffold to facilitate the interaction between MEX3A and PBs proteins. The MEX3A/circMPP6 complex modulates PBs dynamic and promotes UPF-mediated phosphodiesterase 5A (PDE5A) mRNA degradation, consequently leading to the aggressive properties of CRC cells. Clinically, CRC patients exhibiting high MEX3A expression and low PDE5A expression have the poorest overall survival. Our findings reveal a collaboration between MEX3A and circMPP6 in the regulation of mRNA decay through triggering the PBs aggregation, which provides prognostic markers and/or therapeutic targets for CRC.

LIN28B induced PCAT5 promotes endometrial cancer progression and glycolysis via IGF2BP3 deubiquitination.

Endometrial cancer (EC) cells exhibit abnormal glucose metabolism, characterized by increased aerobic glycolysis and decreased oxidative phosphorylation. Targeting cellular glucose metabolism in these cells could be an effective therapeutic approach for EC. This study aimed to assess the roles of LIN28B, PCAT5, and IGF2BP3 in the glucose metabolism, proliferation, migration, and invasion of EC cells. LIN28B highly expressed in EC, binds and stabilizes PCAT5. PCAT5, overexpressed in EC, and its 1485-2288nt region can bind to the KH1-2 domain of IGF2BP3 to prevent MKRN2 from binding to the K294 ubiquitination site of IGF2BP3, thus stabilizing IGF2BP3. Finally, IGF2BP3 promotes the aerobic glycolysis, proliferation, migration and invasion of EC cells by stabilizing the key enzymes of glucose metabolism HK2 and PKM2. Taken together, our data reveal that the LIN28B/PCAT5/IGF2BP3 axis is critical for glucose reprogramming and malignant biological behavior in EC cells. Therefore, targeting this axis may contribute to the development of a novel therapeutic strategy for EC metabolism.

Systematic analysis of RNA-binding proteins identifies targetable therapeutic vulnerabilities in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor with a strong tendency to metastasize, limiting the prognosis of affected patients. Genomic, epigenomic and transcriptomic analyses have demonstrated the exquisite molecular complexity of this tumor, but have not sufficiently defined the underlying mechanisms or identified promising therapeutic targets. To systematically explore RNA-protein interactions relevant to OS, we define the RNA interactomes together with the full proteome and the transcriptome of cells from five malignant bone tumors (four osteosarcomata and one malignant giant cell tumor of the bone) and from normal mesenchymal stem cells and osteoblasts. These analyses uncover both systematic changes of the RNA-binding activities of defined RNA-binding proteins common to all osteosarcomata and individual alterations that are observed in only a subset of tumors. Functional analyses reveal a particular vulnerability of these tumors to translation inhibition and a positive feedback loop involving the RBP IGF2BP3 and the transcription factor Myc which affects cellular translation and OS cell viability. Our results thus provide insight into potentially clinically relevant RNA-binding protein-dependent mechanisms of osteosarcoma.

An Intricate Network Involving the Argonaute ALG-1 Modulates Organismal Resistance to Oxidative Stress.

Cellular response to redox imbalance is crucial for organismal health. microRNAs are implicated in stress responses. ALG-1, the C. elegans ortholog of human AGO2, plays an essential role in microRNA processing and function. Here we investigated the mechanisms governing ALG-1 expression in C. elegans and the players controlling lifespan and stress resistance downstream of ALG-1. We show that upregulation of ALG-1 is a shared feature in conditions linked to increased longevity (e.g., germline-deficient glp-1 mutants). ALG-1 knockdown reduces lifespan and oxidative stress resistance, while overexpression enhances survival against pro-oxidant agents but not heat or reductive stress. R02D3.7 represses alg-1 expression, impacting oxidative stress resistance at least in part via ALG-1. microRNAs upregulated in glp-1 mutants (miR-87-3p, miR-230-3p, and miR-235-3p) can target genes in the protein disulfide isomerase pathway and protect against oxidative stress. This study unveils a tightly regulated network involving transcription factors and microRNAs which controls organisms' ability to withstand oxidative stress.

Proinflammatory cytokines suppress nonsense-mediated RNA decay to impair regulated transcript isoform processing in pancreatic ß cells.

Introduction: Proinflammatory cytokines are implicated in pancreatic ß cell failure in type 1 and type 2 diabetes and are known to stimulate alternative RNA splicing and the expression of nonsense-mediated RNA decay (NMD) components. Here, we investigate whether cytokines regulate NMD activity and identify transcript isoforms targeted in ß cells. Methods: A luciferase-based NMD reporter transiently expressed in rat INS1(832/13), human-derived EndoC-ßH3, or dispersed human islet cells is used to examine the effect of proinflammatory cytokines (Cyt) on NMD activity. The gain- or loss-of-function of two key NMD components, UPF3B and UPF2, is used to reveal the effect of cytokines on cell viability and function. RNA-sequencing and siRNA-mediated silencing are deployed using standard techniques. Results: Cyt attenuate NMD activity in insulin-producing cell lines and primary human ß cells. These effects are found to involve ER stress and are associated with the downregulation of UPF3B. Increases or decreases in NMD activity achieved by UPF3B overexpression (OE) or UPF2 silencing raise or lower Cyt-induced cell death, respectively, in EndoC-ßH3 cells and are associated with decreased or increased insulin content, respectively. No effects of these manipulations are observed on glucose-stimulated insulin secretion. Transcriptomic analysis reveals that Cyt increases alternative splicing (AS)-induced exon skipping in the transcript isoforms, and this is potentiated by UPF2 silencing. Gene enrichment analysis identifies transcripts regulated by UPF2 silencing whose proteins are localized and/or functional in the extracellular matrix (ECM), including the serine protease inhibitor SERPINA1/α-1-antitrypsin, whose silencing sensitizes ß-cells to Cyt cytotoxicity. Cytokines suppress NMD activity via UPR signaling, potentially serving as a protective response against Cyt-induced NMD component expression. Conclusion: Our findings highlight the central importance of RNA turnover in ß cell responses to inflammatory stress.

Plasma metadherin mRNA expression in bladder cancer.

Urinary bladder cancer (BC) is the ninth most common cancer worldwide. At present, the clinical diagnosis of BC depends on self-reported symptoms, tissue biopsy specimens by cystoscopy and from voided urine cytology. However, cystoscopy is an invasive examination and voided urine cytology has low sensitivity, which might provoke misdiagnosis. The search for cancer biomarkers in blood is worthy of intense attention due to patients' comfort and ease of sampling. This work aimed to study expression of mRNA metadherin (MTDH) in plasma, serum BC specific antigen 1 (BLCA-1) and cystatin C as biomarkers of BC and their relation to different disease stages. This study included 59 BC patients, 11 patients with benign bladder lesion and 18 subjects as normal controls. MTDH expression was assessed by real time polymerase chain reaction, BLCA-1, and cystatin C by the enzyme linked immunosorbent assay. The three biomarkers were elevated in BC patients than patients with benign bladder diseases and controls. Patients with BC grade 3 and 4 had higher cystatin C, BLCA-1 and MTDH in comparison to patients with grade 1 and grade 2 (p=0.000). The receiver operating characteristic curve analysis showed that BLCA-1 at a cutoff point of 32.5 ng/ml and area under the curve of 1.00, had 100% accuracy, 100% sensitivity, 100% specificity, 100% positive predictive values and 100% negative predictive value. In conclusion, BLCA-1 was a better biomarker than cystatin C and MTDH. Cystatin C, BLCA-1 and MTDH levels, can differentiate between benign bladder lesion and BC and correlated with tumor grades.especially with OL-HDF compared to HF-HD, with acceptable albumin loss in the dialysate.

RPL9 acts as an oncogene by shuttling miRNAs through exosomes in human hepatocellular carcinoma cells.

The exosomal pathway is an essential mechanism that regulates the abnormal content of microRNAs (miRNAs) in hepatocellular carcinoma (HCC). The directional transport of miRNAs requires the assistance of RNA­binding proteins (RBPs). The present study found that RBPs participate in the regulation of miRNA content through the exosomal pathway in HCC cells. First, differential protein expression profiles in the serum exosomes of patients with HCC and benign liver disease were detected using mass spectrometry. The results revealed that ribosomal protein L9 (RPL9) was highly expressed in serum exosomes of patients with HCC. In addition, the downregulation of RPL9 markedly suppressed the proliferation, migration and invasion of HCC cells and reduced the biological activity of HCC­derived exosomes. In addition, using miRNA microarrays, the changes in exosomal miRNA profiles in HCC cells caused by RPL9 knockdown were examined. miR­24­3p and miR­185­5p were most differentially expressed, as verified by reverse transcription­quantitative PCR. Additionally, using RNA immunoprecipitation, it was found that RPL9 was directly bound to the two miRNAs and immunofluorescence assays confirmed that RPL9 was able to carry miRNAs into recipient cells via exosomes. Overexpression of miR­24­3p in cells increased the accumulation of miR­24­3p in exosomes and simultaneously upregulated RPL9. Excessive expression of miR­24­3p in exosomes also increased their bioactivity. Exosome­mediated miRNA regulation and transfer require the involvement of RBPs. RPL9 functions as an oncogene, can directly bind to specific miRNAs and can be co­transported to receptor cells through exosomes, thereby exerting its biological functions. These findings provide a novel approach for modulating miRNA profiles in HCC.

The RNA-binding protein RBMS3 inhibits the progression of colon cancer by regulating the stability of LIMS1 mRNA.

BACKGROUND: The RNA-binding motif single-stranded interacting protein 3 (RBMS3) is a constituent of the RNA-binding motif (RBM) protein family, which assumes a pivotal role in governing cellular biogenesis processes such as the cell cycle and apoptosis. Despite an abundance of studies elucidating RBMS3's divergent roles in the genesis and advancement of various tumors, its involvement in colon cancer remains enigmatic. METHODS: The present investigation employed data analysis from TCGA and GTEx to unveil that RBMS3 expression demonstrated a diminished presence in colon cancer tissues when juxtaposed with normal colon tissues. The effect of RBMS3 and LIM zinc finger domain 1 (LIMS1) on colon cancer was substantiated via animal models and cellular experiments. The connection between RBMS3 and LIM zinc finger domain 1 (LIMS1) was verified by molecular biology methods. RESULTS: The study conclusively ascertained that augmenting RBMS3 expression quells the proliferation, migration, and invasion of colon cancer cells. Furthermore, the inquiry unveiled a plausible mechanism through which RBMS3 impacts the expression of LIMS1 by modulating its mRNA stability. The investigation ascertained that RBMS3 inhibits the progression of colon cancer by regulating LIMS1. The inhibitory function of LIMS1 and RBMS3 is closely intertwined in colon cancer, with knocking down LIMS1 being able to rescue the inhibitory effect of RBMS3 overexpression on the functionality of colon cancer cell CONCLUSIONS: The discernments delineate RBMS3 as a novel suppressor of cancer via LIMS1, thereby bestowing fresh therapeutic possibilities and illuminating the intricacies of colon cancer.

CircPRMT5 promotes progression of osteosarcoma by recruiting CNBP to regulate the translation and stability of CDK6 mRNA.

Research has demonstrated that circular RNAs (circRNAs) exert critical functions in the occurrence and progression of numerous malignant tumors. CircPRMT5 was recently reported to be involved in the pathogenesis of cancers. However, the potential role of circPRMT5 in osteosarcoma needs further investigation. In present study, our results suggested that circPRMT5 was highly upregulated in osteosarcoma cells and mainly localizes in the cytoplasm. CircPRMT5 promoted the proliferation, migration and invasion capacities of osteosarcoma cells, and suppressed cell apoptosis. Knockdown of circPRMT5 exerted the opposite effects. Mechanically, circPRMT5 promoted the binding of CNBP to CDK6 mRNA, which enhanced the stability of CDK6 mRNA and facilitated its translation, thereby promoting the progression of osteosarcoma. Knockdown of CDK6 reversed the promoting effect of circPRMT5 on osteosarcoma cells. These findings suggest that circPRMT5 promotes osteosarcoma cell malignant activity by recruiting CNBP to regulate the translation and stability of CDK6 mRNA. Thus, circPRMT5 may represent a promising therapeutic target for osteosarcoma.

Divergence in regulatory mechanisms of GR-RBP genes in different plants under abiotic stress.

The IVa subfamily of glycine-rich proteins (GRPs) comprises a group of glycine-rich RNA binding proteins referred to as GR-RBPa here. Previous studies have demonstrated functions of GR-RBPa proteins in regulating stress response in plants. However, the mechanisms responsible for the differential regulatory functions of GR-RBPa proteins in different plant species have not been fully elucidated. In this study, we identified and comprehensively studied a total of 34 GR-RBPa proteins from five plant species. Our analysis revealed that GR-RBPa proteins were further classified into two branches, with proteins in branch I being relatively more conserved than those in branch II. When subjected to identical stresses, these genes exhibited intensive and differential expression regulation in different plant species, corresponding to the enrichment of cis-acting regulatory elements involving in environmental and internal signaling in these genes. Unexpectedly, all GR-RBPa genes in branch I underwent intensive alternative splicing (AS) regulation, while almost all genes in branch II were only constitutively spliced, despite having more introns. This study highlights the complex and divergent regulations of a group of conserved RNA binding proteins in different plants when exposed to identical stress conditions. These species-specific regulations may have implications for stress responses and adaptations in different plant species.

circTADA2A inhibited SLC38A1 expression and suppresses melanoma progression through the prevention of CNBP trans-activation.

BACKGROUND: CircTADA2A has been demonstrated to play critical roles in the occurrence and development of human cancer. However, the expression pattern and biological mechanisms of circTADA2A in melanoma remains largely unknown. METHODS: CircTADA2A were detected by quantitative real-time RT-PCR (qRT-PCR) and validated by Sanger sequencing. Function of circTADA2A and its protein partner in melanoma cells was investigated using RNA interference and overexpression assays. Interaction of circTADA2A, CCHC-type zinc finger nucleic acid binding protein (CNBP) and solute carrier family 38 member 1 (SLC38A1) was confirmed by RNA immunoprecipitation, RNA pull-down, and dual-luciferase reporter assay. The expression of genes and proteins were detected by qRT-PCR and western blot assays. RESULTS: Data from the investigation showed that a novel circRNA (circTADA2A, hsa_circ_0043278) was markedly downregulated in melanoma cells. Functionally, circTADA2A repressed cell proliferation, migration, invasion in melanoma cells. Mechanistically, circTADA2A interacted with CNBP, acting to suppress the binding of CNBP to the SLC38A1 promoter and subsequently restrained SLC38A1 transcription, which resulting in repression of melanoma progression. CONCLUSIONS: CircTADA2A suppresses melanoma progression by regulating CNBP/SLC38A1 axis, indicating a potential therapeutic target in melanoma.

USP7 promotes cervical cancer progression by stabilizing MTDH expression through deubiquitination.

BACKGROUND: Metadherin (MTDH) and ubiquitin specific protease 7 (USP7) have been identified to involve in the tumorigenesis of cervical cancer (CC). USP7 is one of the deubiquitinating enzymes. Here, this study aimed to explore whether USP7 affected CC progression via interacting with MTDH and regulating its stability via deubiquitination. METHODS: qRT-PCR and western blotting assays detected the levels of genes and proteins. Functional analysis was conducted using 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, and tube formation assays, respectively. Proteins between USP7 and MTDH were identified by co-immunoprecipitation assay. A mouse xenograft model was established for in vivo analysis. RESULTS: MTDH was highly expressed in CC tissues and cells, silencing of MTDH suppressed CC cell proliferation, migration, invasion, angiogenesis, and macrophage M2 polarization. Mechanistically, USP7 directly bound to MTDH, and maintained its stability by removing ubiquitination on MTDH. CC tissues and cells showed high USP7 expression, and USP7 knockdown also inhibited CC cell proliferation, migration, invasion, angiogenesis and macrophage M2 polarization, and these effects mediated by USP7 knockdown were reversed by MTDH overexpression. Moreover, USP7 knockdown impeded CC growth in vivo by regulating MTDH. CONCLUSION: Collectively, USP7 promoted CC cell proliferation, migration, invasion, angiogenesis, and macrophage M2 polarization in vitro, as well as tumor growth in vivo by regulating MTDH.

RBM10 C761Y mutation induced oncogenic ASPM isoforms and regulated ß-catenin signaling in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) comprises a heterogeneous group of biliary tract cancer. Our previous CCA mutation pattern study focused on genes in the post-transcription modification process, among which the alternative splicing factor RBM10 captured our attention. However, the roles of RBM10 wild type and mutations in CCA remain unclear. METHODS: RBM10 mutation spectrum in CCA was clarified using our initial data and other CCA genomic datasets from domestic and international sources. Real-time PCR and tissue microarray were used to detect RBM10 clinical association. Function assays were conducted to investigate the effects of RBM10 wild type and mutations on CCA. RNA sequencing was to investigate the changes in alternative splicing events in the mutation group compared to the wild-type group. Minigene splicing reporter and interaction assays were performed to elucidate the mechanism of mutation influence on alternative splicing events. RESULTS: RBM10 mutations were more common in Chinese CCA populations and exhibited more protein truncation variants. RBM10 exerted a tumor suppressive effect in CCA and correlated with favorable prognosis of CCA patients. The overexpression of wild-type RBM10 enhanced the ASPM exon18 exon skipping event interacting with SRSF2. The C761Y mutation in the C2H2-type zinc finger domain impaired its interaction with SRSF2, resulting in a loss-of-function mutation. Elevated ASPM203 stabilized DVL2 and enhanced ß-catenin signaling, which promoted CCA progression. CONCLUSIONS: Our results showed that RBM10C761Y-modulated ASPM203 promoted CCA progression in a Wnt/ß-catenin signaling-dependent manner. This study may enhance the understanding of the regulatory mechanisms that link mutation-altering splicing variants to CCA.

Metadherin promotes stem cell phenotypes and correlated with immune infiltration in hepatocellular carcinoma.

BACKGROUND: Metadherin (MTDH) is a key oncogene in most cancer types, including hepatocellular carcinoma (HCC). Notably, MTDH does not affect the stemness pheno-type or immune infiltration of HCC. AIM: To explore the role of MTDH on stemness and immune infiltration in HCC. METHODS: MTDH expression in HCC tissues was detected using TCGA and GEO databases. Immunohistochemistry was used to analyze the tissue samples. MTDH was stably knocked down or overexpressed by lentiviral transfection in the two HCC cell lines. The invasion and migration abilities of HCC cells were evaluated using Matrigel invasion and wound healing assays. Next, we obtained liver cancer stem cells from the spheroids by culturing them in a serum-free medium. Gene expression was determined by western blotting and quantitative reverse transcri-ption PCR. Flow cytometry, immunofluorescence, and tumor sphere formation assays were used to characterize stem-like cells. The effects of MTDH inhibition on tumor growth were evaluated in vivo. The correlation of MTDH with immune cells, immunomodulators, and chemokines was analyzed using ssGSEA and TISIDB databases. RESULTS: HCC tissues expressed higher levels of MTDH than normal liver tissues. High MTDH expression was associated with a poor prognosis. HCC cells overexpressing MTDH exhibited stronger invasion and migration abilities, exhibited a stem cell-like phenotype, and formed spheres; however, MTDH inhibition attenuated these effects. MTDH inhibition suppressed HCC progression and CD133 expression in vivo. MTDH was positively correlated with immature dendritic, T helper 2 cells, central memory CD8+ T, memory B, activated dendritic, natural killer (NK) T, NK, activated CD4+ T, and central memory CD4+ T cells. MTDH was negatively correlated with activated CD8+ T cells, eosinophils, activated B cells, monocytes, macrophages, and mast cells. A positive correlation was observed between the MTDH level and CXCL2 expression, whereas a negative correlation was observed between the MTDH level and CX3CL1 and CXCL12 expression. CONCLUSION: High levels of MTDH expression in patients with HCC are associated with poor prognosis, promoting tumor stemness, immune infiltration, and HCC progression.

Depletion of the N6-Methyladenosine (m6A) reader protein IGF2BP3 induces ferroptosis in glioma by modulating the expression of GPX4.

Emerging evidence highlights the multifaceted contributions of m6A modifications to glioma. IGF2BP3, a m6A modification reader protein, plays a crucial role in post-transcriptional gene regulation. Though several studies have identified IGF2BP3 as a poor prognostic marker in glioma, the underlying mechanism remains unclear. In this study, we demonstrated that IGF2BP3 knockdown is detrimental to cell growth and survival in glioma cells. Notably, we discovered that IGF2BP3 regulated ferroptosis by modulating the protein expression level of GPX4 through direct binding to a specific motif on GPX4 mRNA. Strikingly, the m6A modification at this motif was found to be critical for GPX4 mRNA stability and translation. Furthermore, IGF2BP3 knockdown glioma cells were incapable of forming tumors in a mouse xenograft model and were more susceptible to phagocytosis by microglia. Our findings shed light on an unrecognized regulatory function of IGF2BP3 in ferroptosis. The identification of a critical m6A site within the GPX4 transcript elucidates the significance of post-transcriptional control in ferroptosis.