Alternative splicing of a chromatin modifier alters the transcriptional regulatory programs of stem cell maintenance and neuronal differentiation.

Development of embryonic stem cells (ESCs) into neurons requires intricate regulation of transcription, splicing, and translation, but how these processes interconnect is not understood. We found that polypyrimidine tract binding protein 1 (PTBP1) controls splicing of DPF2, a subunit of BRG1/BRM-associated factor (BAF) chromatin remodeling complexes. Dpf2 exon 7 splicing is inhibited by PTBP1 to produce the DPF2-S isoform early in development. During neuronal differentiation, loss of PTBP1 allows exon 7 inclusion and DPF2-L expression. Different cellular phenotypes and gene expression programs were induced by these alternative DPF2 isoforms. We identified chromatin binding sites enriched for each DPF2 isoform, as well as sites bound by both. In ESC, DPF2-S preferential sites were bound by pluripotency factors. In neuronal progenitors, DPF2-S sites were bound by nuclear factor I (NFI), while DPF2-L sites were bound by CCCTC-binding factor (CTCF). DPF2-S sites exhibited enhancer modifications, while DPF2-L sites showed promoter modifications. Thus, alternative splicing redirects BAF complex targeting to impact chromatin organization during neuronal development.

MAPP unravels frequent co-regulation of splicing and polyadenylation by RNA-binding proteins and their dysregulation in cancer.

Maturation of eukaryotic pre-mRNAs via splicing and polyadenylation is modulated across cell types and conditions by a variety of RNA-binding proteins (RBPs). Although there exist over 1,500 RBPs in human cells, their binding motifs and functions still remain to be elucidated, especially in the complex environment of tissues and in the context of diseases. To overcome the lack of methods for the systematic and automated detection of sequence motif-guided pre-mRNA processing regulation from RNA sequencing (RNA-Seq) data we have developed MAPP (Motif Activity on Pre-mRNA Processing). Applying MAPP to RBP knock-down experiments reveals that many RBPs regulate both splicing and polyadenylation of nascent transcripts by acting on similar sequence motifs. MAPP not only infers these sequence motifs, but also unravels the position-dependent impact of the RBPs on pre-mRNA processing. Interestingly, all investigated RBPs that act on both splicing and 3' end processing exhibit a consistently repressive or activating effect on both processes, providing a first glimpse on the underlying mechanism. Applying MAPP to normal and malignant brain tissue samples unveils that the motifs bound by the PTBP1 and RBFOX RBPs coordinately drive the oncogenic splicing program active in glioblastomas demonstrating that MAPP paves the way for characterizing pre-mRNA processing regulators under physiological and pathological conditions.

The Regulatory Network of hnRNPs Underlying Regulating PKM Alternative Splicing in Tumor Progression.

One of the hallmarks of cancer is metabolic reprogramming in tumor cells, and aerobic glycolysis is the primary mechanism by which glucose is quickly transformed into lactate. As one of the primary rate-limiting enzymes, pyruvate kinase (PK) M is engaged in the last phase of aerobic glycolysis. Alternative splicing is a crucial mechanism for protein diversity, and it promotes PKM precursor mRNA splicing to produce PKM2 dominance, resulting in low PKM1 expression. Specific splicing isoforms are produced in various tissues or illness situations, and the post-translational modifications are linked to numerous disorders, including cancers. hnRNPs are one of the main components of the splicing factor families. However, there have been no comprehensive studies on hnRNPs regulating PKM alternative splicing. Therefore, this review focuses on the regulatory network of hnRNPs on PKM pre-mRNA alternative splicing in tumors and clinical drug research. We elucidate the role of alternative splicing in tumor progression, prognosis, and the potential mechanism of abnormal RNA splicing. We also summarize the drug targets retarding tumorous splicing events, which may be critical to improving the specificity and effectiveness of current therapeutic interventions.

PCBP1 interacts with the HTLV-1 Tax oncoprotein to potentiate NF-κB activation.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma. The HTLV-1 Tax constitutively activates nuclear factor-κB (NF-κB) to promote the survival and transformation of HTLV-1-infected T cells. Despite extensive study of Tax, how Tax interacts with host factors to regulate NF-κB activation and HTLV-1-driven cell proliferation is not entirely clear. Here, we showed that overexpression of Poly (rC)-binding protein 1 (PCBP1) promoted Tax-mediated IκB kinase (IKK)-NF-κB signaling activation, whereas knockdown of PCBP1 attenuated Tax-dependent IKK-NF-κB activation. However, Tax activation of HTLV-1 long terminal repeat was unaffected by PCBP1. Furthermore, depletion of PCBP1 led to apoptosis and reduced proliferation of HTLV-1-transformed cells. Mechanistically, PCBP1 interacted and co-localized with Tax in the cytoplasm, and PCBP1 KH3 domain was indispensable for the interaction between PCBP1 and Tax. Moreover, PCBP1 facilitated the assembly of Tax/IKK complex. Collectively, our results demonstrated that PCBP1 may exert an essential effect in Tax/IKK complex combination and subsequent NF-κB activation, which provides a novel insight into the pathogenetic mechanisms of HTLV-1.

Inhibition of IGF2BP1 attenuates the progression of endometriosis through PTBP1.

INTRODUCTION: Endometriosis (EMs), manifested by pain and infertility, is a chronic inflammatory disease. The precise pathophysiology of this disease remains uncertain. Insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) and polypyrimidine tract-binding protein 1 (PTBP1) have both been found to regulate proliferation, apoptosis, and invasion. This study aimed to investigate the effects of IGF2BP1/PTBP1 in treating EMs. MATERIALS AND METHODS: qRT-PCR and western blotting were employed to quantify IGF2BP1 and PTBP1 expression in six patients with EMs (mean age 33.83 years). The correlation analysis, STRING database prediction, and RNA immunoprecipitation were utilized to identify the relationship between IGF2BP1 and PTBP1. Ectopic endometrial volume, weight, HE staining, and IGF2BP1 silencing were utilized to estimate the effects of IGF2BP1 in EMs model rats. qRT-PCR, CCK-8, 5-ethynyl-2'-deoxyuridine (EDU) labeling, Transwell assay, and flow cytometry were utilized to assess the effects of IGF2BP1/PTBP1 on the proliferation, migration, invasion, and apoptosis of ectopic endometrial stromal cells (eESCs). Furthermore, western blotting was employed to evaluate expressions of PCNA, VEGF, and E-cadherin in EMs rats and eESCs. RESULTS: The mRNA and protein levels of IGF2BP1 and PTBP1 in the ectopic and eutopic endometrium of EMs patients were significantly increased. RNA immunoprecipitation revealed a close interaction of IGF2BP1 with PTBP1. Additionally, the endometrial volume, weight, and histopathologic scores in rats were significantly reduced after IGF2BP1 silencing. IGF2BP1 silencing also decreased the expression of PCNA and VEGF, and increased E-cadherin expression in endometrial tissues of EMs rats. Moreover, IGF2BP1 silencing inhibited proliferation, migration, and invasion and promoted apoptosis through PTBP1 in eESCs. CONCLUSIONS: IGF2BP1 exhibits potential beneficial properties in the management of EMs by interacting with PTBP1, thereby highlighting IGF2BP1 as a promising therapeutic target for EMs.

KIS counteracts PTBP2 and regulates alternative exon usage in neurons.

Alternative RNA splicing is an essential and dynamic process in neuronal differentiation and synapse maturation, and dysregulation of this process has been associated with neurodegenerative diseases. Recent studies have revealed the importance of RNA-binding proteins in the regulation of neuronal splicing programs. However, the molecular mechanisms involved in the control of these splicing regulators are still unclear. Here, we show that KIS, a kinase upregulated in the developmental brain, imposes a genome-wide alteration in exon usage during neuronal differentiation in mice. KIS contains a protein-recognition domain common to spliceosomal components and phosphorylates PTBP2, counteracting the role of this splicing factor in exon exclusion. At the molecular level, phosphorylation of unstructured domains within PTBP2 causes its dissociation from two co-regulators, Matrin3 and hnRNPM, and hinders the RNA-binding capability of the complex. Furthermore, KIS and PTBP2 display strong and opposing functional interactions in synaptic spine emergence and maturation. Taken together, our data uncover a post-translational control of splicing regulators that link transcriptional and alternative exon usage programs in neuronal development.

The leader RNA of SARS-CoV-2 sequesters polypyrimidine tract binding protein (PTBP1) and influences pre-mRNA splicing in infected cells.

The large amount of viral RNA produced during infections has the potential to interact with and effectively sequester cellular RNA binding proteins, thereby influencing aspects of post-transcriptional gene regulation in the infected cell. Here we demonstrate that the abundant 5' leader RNA region of SARS-CoV-2 viral RNAs can interact with the cellular polypyrimidine tract binding protein (PTBP1). Interestingly, the effect of a knockdown of PTBP1 protein on cellular gene expression is also mimicked during SARS-CoV-2 infection, suggesting that this protein may be functionally sequestered by viral RNAs. Consistent with this model, the alternative splicing of mRNAs that is normally controlled by PTBP1 is dysregulated during SARS-CoV-2 infection. Collectively, these data suggest that the SARS-CoV-2 leader RNA sequesters the cellular PTBP1 protein during infection, resulting in significant impacts on the RNA biology of the host cell. These alterations in post-transcriptional gene regulation may play a role in SARS-CoV-2 mediated molecular pathogenesis.

USP46 enhances tamoxifen resistance in breast cancer cells by stabilizing PTBP1 to facilitate glycolysis.

Tamoxifen (TAM) is the primary drug for treating estrogen receptor alpha-positive (ER+) breast cancer (BC). However, resistance to TAM can develop in some patients, limiting its therapeutic efficacy. The ubiquitin-specific protease (USP) family has been associated with the development, progression, and drug resistance of various cancers. To explore the role of USPs in TAM resistance in BC, we used qRT-PCR to compare USP expression between TAM-sensitive (MCF-7 and T47D) and TAM-resistant cells (MCF-7R and T47DR). We then modulated USP46 expression and examined its impact on cell proliferation, drug resistance (via CCK-8 and EdU experiments), glycolysis levels (using a glycolysis detection assay), protein interactions (confirmed by co-IP), and protein changes (analyzed through Western blotting). Our findings revealed that USP46 was significantly overexpressed in TAM-resistant BC cells, leading to the inhibition of the ubiquitin degradation of polypyrimidine tract-binding protein 1 (PTBP1). Overexpression of PTBP1 increased the PKM2/PKM1 ratio, promoted glycolysis, and intensified TAM resistance in BC cells. Knockdown of USP46 induced downregulation of PTBP1 protein by promoting its K48-linked ubiquitination, resulting in a decreased PKM2/PKM1 ratio, reduced glycolysis, and heightened TAM sensitivity in BC cells. In conclusion, this study highlights the critical role of the USP46/PTBP1/PKM2 axis in TAM resistance in BC. Targeted therapy against USP46 may represent a promising strategy to improve the prognosis of TAM-resistant patients.

PTBP1 promotes the progression of hepatocellular carcinoma by enhancing the oncogenic splicing switch of FGFR2.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer accounting for 90% of cases. It is a highly invasive and deadly cancer with a gradual onset. Polypyrimidine tract-binding protein 1 (PTBP1) is an important RNA-binding protein involved in RNA metabolism and has been linked to oncogenic splicing events. While the oncogenic role of PTBP1 in HCC cells has been established, the exact mechanism of action remains unclear. This study aimed to investigate the functional connection between PTBP1 and dysregulated splicing events in HCC. Through immunoprecipitation-mass spectrometry analyses, we discovered that the proteins bound to PTBP1 were significantly enriched in the complex responsible for the alternative splicing of FGFR2 (fibroblast growth factor receptor 2). Further RNA immunoprecipitation and quantitative PCR assays confirmed that PTBP1 down-regulated the FGFR2-IIIb isoform levels and up-regulated the FGFR2-IIIc isoform levels in HCC cells, leading to a switch from FGFR2-IIIb to FGFR2-IIIc isoforms. Subsequent functional evaluations using CCK-8, transwell, and plate clone formation assays in HCC cell lines HepG2 and Huh7 demonstrated that FGFR2-IIIb exhibited tumor-suppressive effects, while FGFR2-IIIc displayed tumor-promoting effects. In conclusion, this study provides insights into the PTBP1-mediated alternative splicing mechanism in HCC progression, offering a new theoretical basis for the prevention and treatment of this malignancy. Mechanistically, the isoform switch from FGFR2-IIIb to FGFR2-IIIc promoted epithelial-mesenchymal transformation (EMT) of HCC cells and activated the FGFR cascades ERK and AKT pathways.

Aberrant RBMX expression is relevant for cancer prognosis and immunotherapy response.

Cancer accounts for the highest rates of morbidity and mortality worldwide. RNA binding motif protein X-linked (RBMX) is a nuclear RNA-binding protein, associated with certain types of cancer by participating in the integration of sister chromatids and a combination of ribonucleoprotein complexes. However, the specific role of RBMX in cancer immunity remains unknown. This study presents the aberrant expression levels, single-cell distributions, effective prognostic roles, immune cell infiltration associations, and immunotherapy responses of RBMX as a biomarker in various types of cancer. Moreover, it validates the aberrant expression of RBMX in clinical cancer samples. Furthermore, we also evaluated the relationships between RBMX expression and myeloid-derived suppressor cells in clinical samples by immunofluorescent staining. The results showed that knockdown of RBMX can impair the proliferation, migration, and invasion of liver cancer cells. Finally, we indicated that RBMX may play an immunoregulatory role in cancer progression, affecting the therapeutic effects of immune checkpoint inhibitors in patients with cancer.

KLF15 transcriptionally activates LINC00689 to inhibit colorectal cancer development.

Colorectal cancer is a grievous health concern, we have proved long non-coding RNA LINC00689 is considered as a potential diagnosis biomarker for colorectal cancer, and it is necessary to further investigate its upstream and downstream mechanisms. Here, we show that KLF15, a transcription factor, exhibits the reduced expression in colorectal cancer. KLF15 suppresses the proliferative and metastatic capacities of colorectal cancer cells both in vitro and in vivo by transcriptionally activating LINC00689. Subsequently, LINC00689 recruits PTBP1 protein to enhance the stability of LATS2 mRNA in the cytoplasm. This stabilization causes the suppression of the YAP1/ß-catenin pathway and its target downstream genes. Our findings highlight a regulatory network involving KLF15, LINC00689, PTBP1, LATS2, and the YAP1/ß-catenin pathway in colorectal cancer, shedding light on potential therapeutic targets for colorectal cancer therapy.

Heterogenous nuclear ribonucleoprotein D-like controls endothelial cell functions.

HnRNPs are ubiquitously expressed RNA-binding proteins, tightly controlling posttranscriptional gene regulation. Consequently, hnRNP networks are essential for cellular homeostasis and their dysregulation is associated with cancer and other diseases. However, the physiological function of hnRNPs in non-cancerous cell systems are poorly understood. We analyzed the importance of HNRNPDL in endothelial cell functions. Knockdown of HNRNPDL led to impaired proliferation, migration and sprouting of spheroids. Transcriptome analysis identified cyclin D1 (CCND1) and tropomyosin 4 (TPM4) as targets of HNRNPDL, reflecting the phenotypic changes after knockdown. Our findings underline the importance of HNRNPDL for the homeostasis of physiological processes in endothelial cells.

An in vitro-transcribed circular RNA targets the mitochondrial inner membrane cardiolipin to ablate EIF4G2+/PTBP1+ pan-adenocarcinoma.

In vitro-transcribed (IVT) mRNA has arisen as a rapid method for the production of nucleic acid drugs. Here, we have constructed an oncolytic IVT mRNA that utilizes human rhinovirus type 2 (HRV2) internal ribosomal entry sites (IRESs) to selectively trigger translation in cancer cells with high expression of EIF4G2 and PTBP1. The oncolytic effect was provided by a long hGSDMDc .825 T>A/c.884 A>G-F1LCT mutant mRNA sequence with mitochondrial inner membrane cardiolipin targeting toxicity that triggers mitophagy. Utilizing the permuted intron-exon (PIE) splicing circularization strategy and lipid nanoparticle (LNP) encapsulation reduced immunogenicity of the mRNA and enabled delivery to eukaryotic cells in vivo. Engineered HRV2 IRESs-GSDMDp.D275E/E295G-F1LCT circRNA-LNPs (GSDMDENG circRNA) successfully inhibited EIF4G2+/PTBP1+ pan-adenocarcinoma xenografts growth. Importantly, in a spontaneous tumor model with abnormal EIF4G2 and PTBP1 caused by KRAS G12D mutation, GSDMDENG circRNA significantly prevented the occurrence of pancreatic, lung and colon adenocarcinoma, improved the survival rate and induced persistent KRAS G12D tumor antigen-specific cytotoxic T lymphocyte responses.

Gustavson syndrome is caused by an in-frame deletion in RBMX associated with potentially disturbed SH3 domain interactions.

RNA binding motif protein X-linked (RBMX) encodes the heterogeneous nuclear ribonucleoprotein G (hnRNP G) that regulates splicing, sister chromatid cohesion and genome stability. RBMX knock down experiments in various model organisms highlight the gene's importance for brain development. Deletion of the RGG/RG motif in hnRNP G has previously been associated with Shashi syndrome, however involvement of other hnRNP G domains in intellectual disability remain unknown. In the current study, we present the underlying genetic and molecular cause of Gustavson syndrome. Gustavson syndrome was first reported in 1993 in a large Swedish five-generation family presented with profound X-linked intellectual disability and an early death. Extensive genomic analyses of the family revealed hemizygosity for a novel in-frame deletion in RBMX in affected individuals (NM_002139.4; c.484_486del, p.(Pro162del)). Carrier females were asymptomatic and presented with skewed X-chromosome inactivation, indicating silencing of the pathogenic allele. Affected individuals presented minor phenotypic overlap with Shashi syndrome, indicating a different disease-causing mechanism. Investigation of the variant effect in a neuronal cell line (SH-SY5Y) revealed differentially expressed genes enriched for transcription factors involved in RNA polymerase II transcription. Prediction tools and a fluorescence polarization assay imply a novel SH3-binding motif of hnRNP G, and potentially a reduced affinity to SH3 domains caused by the deletion. In conclusion, we present a novel in-frame deletion in RBMX segregating with Gustavson syndrome, leading to disturbed RNA polymerase II transcription, and potentially reduced SH3 binding. The results indicate that disruption of different protein domains affects the severity of RBMX-associated intellectual disabilities.

Therapeutic potential of natural antisense transcripts and various mechanisms involved for clinical applications and disease prevention.

Antisense transcription, a prevalent occurrence in mammalian genomes, gives rise to natural antisense transcripts (NATs) as RNA molecules. These NATs serve as agents of diverse transcriptional and post-transcriptional regulatory mechanisms, playing crucial roles in various biological processes vital for cell function and immune response. However, when their normal functions are disrupted, they can contribute to human diseases. This comprehensive review aims to establish the molecular foundation linking NATs to the development of disorders like cancer, neurodegenerative conditions, and cardiovascular ailments. Additionally, we evaluate the potential of oligonucleotide-based therapies targeting NATs, presenting both their advantages and limitations, while also highlighting the latest advancements in this promising realm of clinical investigation.Abbreviations: NATs- Natural antisense transcripts, PRC1- Polycomb Repressive Complex 1, PRC2- Polycomb Repressive Complex 2, ADARs- Adenosine deaminases acting on RNA, BDNF-AS- Brain-derived neurotrophic factor antisense transcript, ASOs- Antisense oligonucleotides, SINEUPs- Inverted SINEB2 sequence-mediated upregulating molecules, PTBP1- Polypyrimidine tract binding protein-1, HNRNPK- heterogeneous nuclear ribonucleoprotein K, MAPT-AS1- microtubule-associated protein tau antisense 1, KCNQ1OT- (KCNQ1 opposite strand/antisense transcript 1, ERK- extracellular signal-regulated kinase 1, USP14- ubiquitin-specific protease 14, EGF- Epidermal growth factor, LSD1- Lysine Specific Demethylase 1, ANRIL- Antisense Noncoding RNA in the INK4 Locus, BWS- Beckwith-Wiedemann syndrome, VEGFA- Vascular Endothelial Growth component A.

LncRNA MIAT Modulates LPS-Induced Acute Kidney Injury via BECN1-Dependent Autophagy by Interacting with PTBP1.

BACKGROUND: Autophagy plays critical adaptive and nonadaptive roles in the pathogenesis of Sepsis-associated acute kidney injury (Sepsis-AKI). However, it remains unknown whether myocardial infarction associated transcript (MIAT) is involved in the process of autophagy in Sepsis-AKI. This study aimed to explore the exact association between MIAT1 and Beclin 1 (BECN1)-mediated autophagy in Sepsis-AKI in vitro. METHODS: HK-2 (human renal tubular epithelial cell line) cells were stimulated by lipopolysaccharide (LPS) to construct a septic kidney injury cell model in vitro. The relative expression changes of genes or proteins in clinical samples and cells were examined by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot. Cell survival was detected by cell counting kit-8 (CCK-8) and flow cytometry analysis. The production of inflammatory mediators was determined using Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR assays. The interlinked relationship between polypyrimidine tract-binding protein 1 (PTBP1) and MIAT or BECN1 was validated by RNA immunoprecipitation (RIP) and RNA pull-down detections. RESULTS: The expression of MIAT was up-regulated in Sepsis-AKI patients and LPS-stimulated HK-2 cells. Down-regulation of MIAT strikingly lightened LPS-induced cell apoptosis and inflammation, but enhanced cell viability. Evidenced by mechanistic experiments, MIAT silencing was confirmed to activate BECN1-mediated cell autophagy by interacting with PTBP1. Furthermore, the elimination of BECN1 remarkably reversed the antiapoptotic and anti-inflammatory roles mediated by MIAT silencing. CONCLUSIONS: In summary, the experimental data reinforced that MIAT downregulation attenuated LPS-stimulated renal cell inflammatory injury by promoting BECN1-mediated autophagy activation through binding to PTBP1, providing some new insights into the function and mechanism of MIAT in Sepsis-associated acute kidney injury (Sepsis-AKI).

FMRP deficiency leads to multifactorial dysregulation of splicing and mislocalization of MBNL1 to the cytoplasm.

Fragile X syndrome (FXS) is a neurodevelopmental disorder that is often modeled in Fmr1 knockout mice where the RNA-binding protein FMRP is absent. Here, we show that in Fmr1-deficient mice, RNA mis-splicing occurs in several brain regions and peripheral tissues. To assess molecular mechanisms of splicing mis-regulation, we employed N2A cells depleted of Fmr1. In the absence of FMRP, RNA-specific exon skipping events are linked to the splicing factors hnRNPF, PTBP1, and MBNL1. FMRP regulates the translation of Mbnl1 mRNA as well as Mbnl1 RNA auto-splicing. Elevated Mbnl1 auto-splicing in FMRP-deficient cells results in the loss of a nuclear localization signal (NLS)-containing exon. This in turn alters the nucleus-to-cytoplasm ratio of MBNL1. This redistribution of MBNL1 isoforms in Fmr1-deficient cells could result in downstream splicing changes in other RNAs. Indeed, further investigation revealed that splicing disruptions resulting from Fmr1 depletion could be rescued by overexpression of nuclear MBNL1. Altered Mbnl1 auto-splicing also occurs in human FXS postmortem brain. These data suggest that FMRP-controlled translation and RNA processing may cascade into a general dys-regulation of splicing in Fmr1-deficient cells.

lncRNA Helf promotes hepatic inflammation and fibrosis by interacting with PTBP1 to facilitate PIK3R5 mRNA stabilization.

BACKGROUND: Hepatic fibrosis is a common consequence of chronic liver diseases without approved antifibrotic therapies. Long noncoding RNAs (lncRNAs) play an important role in various pathophysiological processes. However, the functions of certain lncRNAs involved in mediating the antifibrotic role remain largely unclear. METHODS: The RNA level of lnc-High Expressed in Liver Fibrosis (Helf) was detected in both mouse and human fibrotic livers. Furthermore, lnc-Helf-silenced mice were treated with carbon tetrachloride (CCl4) or bile duct ligation (BDL) to investigate the function of lnc-Helf in liver fibrosis. RESULTS: We found that lnc-Helf has significantly higher expression in human and mouse fibrotic livers as well as M1 polarized hepatic macrophages (HMs) and activated hepatic stellate cells (HSCs). In vivo studies showed that silencing lnc-Helf by AAV8 vector alleviates CCl4- and BDL-induced hepatic inflammation and fibrosis. Furthermore, in vitro experiments revealed that lnc-Helf promotes HSCs activation and proliferation, as well as HMs M1 polarization and proliferation in the absence or presence of cytokine stimulation. Mechanistically, our data illustrated that lnc-Helf interacts with RNA binding protein PTBP1 to promote its interaction with PIK3R5 mRNA, resulting in increased stability and activating the AKT pathway, thus promoting HSCs and HMs activation and proliferation, which augments hepatic inflammation and fibrosis. CONCLUSION: Our results unveil a lnc-Helf/PTBP1/PIK3R5/AKT feedforward, amplifying signaling that exacerbates the process of hepatic inflammation and fibrosis, thus providing a possible therapeutic strategy for hepatic fibrosis.

Cullin-associated and neddylation-dissociated 1 regulate reprogramming of lipid metabolism through SKP1-Cullin-1-F-boxFBXO11 -mediated heterogeneous nuclear ribonucleoprotein A2/B1 ubiquitination and promote hepatocellular carcinoma.

BACKGROUND: Enhanced de novo lipogenesis is essential for hepatocellular carcinoma (HCC). Abnormally high cullin-associated and neddylation-dissociated 1 (CAND1) expression is associated with poor clinical prognosis in HCC. The SKP1-Cullin-1-F-box (SCF) complex consists of the SKP1, Cullin-1 and F-box proteins (FBPs) and performs multiple functions including adipogenesis. SCF complex was modulated by CAND1, but Whether and how the CAND1 promotes HCC by regulating SCF complex and lipogenesis are unknown. METHODS: HCC samples were used to analyze the correlations between CAND1 expression and clinicopathological characteristics such as survival and prognosis. The in vitro functions of CAND1, FBXO11 and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) were measured by cell proliferation, colony formation and migration assays. The in vivo functions were tested in multiple mouse liver cancer models including patient-derived xenograft (PDX), cell line-derived xenograft and AKT/NRASV12-induced primary liver cancer models. Injections of adeno-associated virus targeting CAND1 (AAV-shCAND1) were performed to evaluate the therapeutic efficacy of targeting CAND1. RNA-Seq and lipidomic assays followed by serial biochemical experiments including mass spectrometry, immunoprecipitation and GST pull-down were performed to dissect the underlying mechanisms. RESULTS: CAND1 promoted the expression of lipid synthesis genes by disrupting SCF complex assembly and lipid accumulation. Furthermore, we identified hnRNPA2B1 as a novel F-box protein 11 (FBXO11)-binding partner. FBXO11 directly bound to hnRNPA2B1 and promoted hnRNPA2B1 ubiquitination and subsequent degradation. Our evaluations of the therapeutic efficacy of AAV-shCAND1 injections confirmed that targeting the CAND1-SCFFBXO11 -hnRNPA2B1A signalling axis was therapeutically effective. CAND1 downregulation significantly reduced the tumour burden in a primary mouse liver cancer model and a PDX model. CONCLUSIONS: Our results highlight that CAND1 is associated with poor prognosis in HCC and regulates lipid metabolic reprogramming by dissociating the SCF complex. Targeting the CAND1-SCFFBXO11 -hnRNPA2B1 axis may be a novel strategy for HCC treatment.

LncRNA CCAT1 facilitates the progression of gastric cancer via PTBP1-mediated glycolysis enhancement.

BACKGROUND: Gastric cancer (GC) is one of the most prevalent malignant tumors of the digestive system. As a hallmark of cancer, energy-related metabolic reprogramming is manipulated by multiple factors, including long non-coding RNAs (lncRNAs). Notably, lncRNA CCAT1 has been identified as a crucial regulator in tumor progression. Nevertheless, the precise molecular mechanisms underlying the involvement of CCAT1 in metabolic reprogramming of GC remain unclear. METHODS: Gain- and loss-of-function experiments were performed to evaluate the roles of CCAT1 in tumorigenesis and glycolysis of GC. Bioinformatics analyses and mechanistic experiments, such as mass spectrometry (MS), RNA-pulldown, and RNA immunoprecipitation (RIP), were employed to reveal the potential interacting protein of CCAT1 and elucidate the regulatory mechanism of CCAT1 in GC glycolysis. Moreover, the nude mice xenograft assay was used to evaluate the effect of CCAT1 on GC cells in vivo. RESULTS: In this study, we identified that CCAT1 expression was significantly elevated in the tissues and plasma exosomes of GC patients, as well as GC cell lines. Functional experiments showed that the knockdown of CCAT1 resulted in a substantial decrease in the proliferation, migration and invasion of GC cells both in vitro and in vivo through decreasing the expression of glycolytic enzymes and glycolytic rate. Conversely, overexpression of CCAT1 exhibited contrasting effects. Mechanistically, CCAT1 interacted with PTBP1 and effectively maintained its stability by inhibiting the ubiquitin-mediated degradation process. As a critical splicing factor, PTBP1 facilitated the transition from PKM1 to PKM2, thereby augmenting the glycolytic activity of GC cells and ultimately fostering the progression of GC. CONCLUSIONS: Our findings demonstrate that CCAT1 plays a significant role in promoting the proliferation, migration, and invasion of GC cells through the PTBP1/PKM2/glycolysis pathway, thus suggesting CCAT1's potential as a biomarker and therapeutic target for GC.