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.

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.

RAVER1 hinders lethal EMT and modulates miR/RISC activity by the control of alternative splicing.

The RAVER1 protein serves as a co-factor in guiding the polypyrimidine tract-binding protein (PTBP)-dependent control of alternative splicing (AS). Whether RAVER1 solely acts in concert with PTBPs and how it affects cancer cell fate remained elusive. Here, we provide the first comprehensive investigation of RAVER1-controlled AS in cancer cell models. This reveals a pro-oncogenic role of RAVER1 in modulating tumor growth and epithelial-mesenchymal-transition (EMT). Splicing analyses and protein-association studies indicate that RAVER1 guides AS in association with other splicing regulators, including PTBPs and SRSFs. In cancer cells, one major function of RAVER1 is the stimulation of proliferation and restriction of apoptosis. This involves the modulation of AS events within the miR/RISC pathway. Disturbance of RAVER1 impairs miR/RISC activity resulting in severely deregulated gene expression, which promotes lethal TGFB-driven EMT. Among others, RAVER1-modulated splicing events affect the insertion of protein interaction modules in factors guiding miR/RISC-dependent gene silencing. Most prominently, in all three human TNRC6 proteins, RAVER1 controls AS of GW-enriched motifs, which are essential for AGO2-binding and the formation of active miR/RISC complexes. We propose, that RAVER1 is a key modulator of AS events in the miR/RISC pathway ensuring proper abundance and composition of miR/RISC effectors. This ensures balanced expression of TGFB signaling effectors and limits TGFB induced lethal EMT.

Long noncoding RNA KCNQ1OT1 aggravates cerebral infarction by regulating PTBT1/SIRT1 via miR-16-5p.

Cerebral infarction (CI) is one of the leading causes of disability and death. LncRNAs are key factors in CI progression. Herein, we studied the function of long noncoding RNA KCNQ1OT1 in CI patient plasma samples and in CI models. Quantitative real-time PCR and Western blotting tested gene and protein expressions. The interactions of KCNQ1OT1/PTBP1 and miR-16-5p were analyzed using dual-luciferase reporter and RNA immunoprecipitation assays; MTT assays measured cell viability. Cell migration and angiogenesis were tested by wound healing and tube formation assays. Pathological changes were analyzed by triphenyltetrazolium chloride and routine staining. We found that KCNQ1OT1 and PTBP1 were overexpressed and miR-16-5p was downregulated in CI patient plasma and in oxygen-glucose deprived (OGD) induced mouse brain microvascular endothelial (bEnd.3) cells. KCNQ1OT1 knockdown suppressed pro-inflammatory cytokine production and stimulated angiogenic responses in OGD-bEnd.3 cells. KCNQ1OT1 upregulated PTBP1 by sponging miR-16-5p. PTBP1 overexpression or miR-16-5p inhibition attenuated the effects of KCNQ1OT1 knockdown. PTBP1 silencing protected against OGD-bEnd.3 cell injury by enhancing SIRT1. KCNQ1OT1 silencing or miR-16-5p overexpression also alleviated ischemic injury in a mice middle cerebral artery occlusion model. Thus, KCNQ1OT1 silencing alleviates CI by regulating the miR-16-5p/PTBP1/SIRT1 pathway, providing a theoretical basis for novel therapeutic strategies targeting CI.

CircSEPT9 promotes breast cancer progression by regulating PTBP3 expression via sponging miR-625-5p.

BACKGROUND: Breast cancer (BC) is a common malignancy which threatens the health of women. Circular RNAs (circRNAs) are critical factors in multiple cancers, including BC. The aim of this experiment was to investigate the molecular mechanisms of circRNA Septin 9 (circSEPT9) in the progression of BC. METHODS: CircSEPT9, microRNA-625-5p (miR-625-5p) and polypyrimidine tract-binding protein 3 (PTBP3) levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was performed to detect the protein levels of PTBP3, E-cadherin and vimentin. Cell counting kit-8 assay (CCK8) and thymidine analog 5-ethynyl-2'-deoxyuridine (EDU) was utilized for proliferation examination. Flow cytometry was conducted to measure apoptosis. Transwell assay and wound healing assay to investigate the migration of BC cells. Glucose uptake and lactate production were determined by specific kits. Additionally, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were utilized to verify the interaction. A murine xenograft model was established to investigate the function of circSEPT9 in BC in vivo. RESULTS: Overexpression of circSEPT9 was found in BC tissues and cells. Silencing circSEPT9 impeded BC cell proliferation, migration, epithelial-mesenchymal transition (EMT) and glycolytic metabolism but facilitated cell apoptosis in vitro. Meanwhile, circSEPT9 knockdown constrained tumor growth in vivo. MiR-625-5p was targeted by circSEPT9. The influence of silencing circSEPT9 on BC cell function was regained by miR-625-5p inhibitor. Furthermore, miR-625-5p regulated BC cell malignant phenotypes via downregulating PTBP3. CONCLUSION: circSEPT9 contributed to the malignant progression of BC by up-regulating PTBP3 via sponging miR-625-5p.

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.

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.

RNA Binding Protein PTBP1 Promotes the Metastasis of Gastric Cancer by Stabilizing PGK1 mRNA.

Gastric cancer (GC) is the most common type of malignant tumor within the gastrointestinal tract, and GC metastasis is associated with poor prognosis. Polypyrimidine tract binding protein 1 (PTBP1) is an RNA-binding protein implicated in various types of tumor development and metastasis. However, the role of PTBP1 in GC metastasis remains elusive. In this study, we verified that PTBP1 was upregulated in GC tissues and cell lines, and higher PTBP1 level was associated with poorer prognosis. It was shown that PTBP1 knockdown in vitro inhibited GC cell migration, whereas PTBP1 overexpression promoted the migration of GC cells. In vivo, the knockdown of PTBP1 notably reduced both the size and occurrence of metastatic nodules in a nude mice liver metastasis model. We identified phosphoglycerate kinase 1 (PGK1) as a downstream target of PTBP1 and found that PTBP1 increased the stability of PGK1 by directly binding to its mRNA. Furthermore, the PGK1/SNAIL axis could be required for PTBP1's function in the promotion of GC cell migration. These discoveries suggest that PTBP1 could be a promising therapeutic target for GC.

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.

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.

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.

Tuberculosis-targeted next-generation sequencing and machine learning: An ultrasensitive diagnostic strategy for paucibacillary pulmonary tuberculosis and tuberculous meningitis.

BACKGROUND: Existing diagnostic approaches for paucibacillary tuberculosis (TB) are limited by the low sensitivity of testing methods and difficulty in obtaining suitable samples. METHODS: An ultrasensitive TB diagnostic strategy was established, integrating efficient and specific TB targeted next-generation sequencing and machine learning models, and validated in clinical cohorts to test plasma cfDNA, cerebrospinal fluid (CSF) DNA collected from tuberculous meningitis (TBM) and pediatric pulmonary TB (PPTB) patients. RESULTS: In the detection of 227 samples, application of the specific thresholds of CSF DNA (AUC = 0.974) and plasma cfDNA (AUC = 0.908) yielded sensitivity of 97.01 % and the specificity of 95.65 % in CSF samples and sensitivity of 82.61 % and specificity of 86.36 % in plasma samples, respectively. In the analysis of 44 paired samples from TBM patients, our strategy had a high concordance of 90.91 % (40/44) in plasma cfDNA and CSF DNA with both sensitivity of 95.45 % (42/44). In the PPTB patient, the sensitivity of the TB diagnostic strategy yielded higher sensitivity on plasma specimen than Xpert assay on gastric lavage (28.57 % VS. 15.38 %). CONCLUSIONS: Our TB diagnostic strategy provides greater detection sensitivity for paucibacillary TB, while plasma cfDNA as an easily collected specimen, could be an appropriate sample type for PTB and TBM diagnosis.

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.

PTBP2 attenuation facilitates fibroblast to neuron conversion by promoting alternative splicing of neuronal genes.

The direct conversion of human skin fibroblasts to neurons has a low efficiency and unclear mechanism. Here, we show that the knockdown of PTBP2 significantly enhanced the transdifferentiation induced by ASCL1, MIR9/9∗-124, and p53 shRNA (AMp) to generate mostly GABAergic neurons. Longitudinal RNA sequencing analyses identified the continuous induction of many RNA splicing regulators. Among these, the knockdown of RBFOX3 (NeuN), significantly abrogated the transdifferentiation. Overexpression of RBFOX3 significantly enhanced the conversion induced by AMp; the enhancement was occluded by PTBP2 knockdown. We found that PTBP2 attenuation significantly favored neuron-specific alternative splicing (AS) of many genes involved in synaptic transmission, signal transduction, and axon formation. RBFOX3 knockdown significantly reversed the effect, while RBFOX3 overexpression occluded the enhancement. The study reveals the critical role of neuron-specific AS in the direct conversion of human skin fibroblasts to neurons by showing that PTBP2 attenuation enhances this mechanism in concert with RBFOX3.

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.

The lncRNA MIR99AHG directs alternative splicing of SMARCA1 by PTBP1 to enable invadopodia formation in colorectal cancer cells.

Alternative splicing regulates gene expression and functional diversity and is often dysregulated in human cancers. Here, we discovered that the long noncoding RNA (lncRNA) MIR99AHG regulated alternative splicing to alter the activity of a chromatin remodeler and promote metastatic behaviors in colorectal cancer (CRC). MIR99AHG was abundant in invasive CRC cells and metastatic tumors from patients and promoted motility and invasion in cultured CRC cells. MIR99AHG bound to and stabilized the RNA splicing factor PTBP1, and this complex increased cassette exon inclusion in the mRNA encoding the chromatin remodeling gene SMARCA1. Specifically, MIR99AHG altered the nature of PTBP1 binding to the splice sites on intron 12 of SMARCA1 pre-mRNA, thereby triggering a splicing switch from skipping to including exon 13 to produce the long isoform, SMARCA1-L. SMARCA1, but not SMARCA1-L, suppressed invadopodia formation, cell migration, and invasion. Analysis of CRC samples revealed that the abundance of MIR99AHG transcript positively correlated with that of SMARCA1-L mRNA and PTBP1 protein and with poor prognosis in patients with CRC. Furthermore, TGF-ß1 secretion from cancer-associated fibroblasts increased MIR99AHG expression in CRC cells. Our findings identify an lncRNA that is induced by cues from the tumor microenvironment and that interacts with PTBP1 to regulate alternative splicing, potentially providing a therapeutic target and predictive biomarker for metastatic CRC.