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.

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.

Unstructured linker regions play a role in the differential splicing activities of paralogous RNA binding proteins PTBP1 and PTBP2.

RNA Binding Proteins regulate, in part, alternative pre-mRNA splicing and, in turn, gene expression patterns. Polypyrimidine tract binding proteins PTBP1 and PTBP2 are paralogous RNA binding proteins sharing 74% amino acid sequence identity. Both proteins contain four structured RNA-recognition motifs (RRMs) connected by linker regions and an N-terminal region. Despite their similarities, the paralogs have distinct tissue-specific expression patterns and can regulate discrete sets of target exons. How two highly structurally similar proteins can exert different splicing outcomes is not well understood. Previous studies revealed that PTBP2 is post-translationally phosphorylated in the unstructured N-terminal, Linker 1, and Linker 2 regions that share less sequence identity with PTBP1 signifying a role for these regions in dictating the paralog's distinct splicing activities. To this end, we conducted bioinformatics analysis to determine the evolutionary conservation of RRMs versus linker regions in PTBP1 and PTBP2 across species. To determine the role of PTBP2 unstructured regions in splicing activity, we created hybrid PTBP1-PTBP2 constructs that had counterpart PTBP1 regions swapped to an otherwise PTBP2 protein and assayed on differentially regulated exons. We also conducted molecular dynamics studies to investigate how negative charges introduced by phosphorylation in PTBP2 unstructured regions can alter their physical properties. Collectively, results from our studies reveal an important role for PTBP2 unstructured regions and suggest a role for phosphorylation in the differential splicing activities of the paralogs on certain regulated exons.

[RBMX overexpression inhibits proliferation, migration, invasion and glycolysis of human bladder cancer cells by downregulating PKM2].

OBJECTIVE: To investigate the role of RNA-binding motif protein X-linked (RBMX) in regulating the proliferation, migration, invasion and glycolysis in human bladder cancer cells. METHODS: A lentivirus vectors system and RNA interference technique were used to construct bladder cancer 1376 and UC-3 cell models with RBMX overexpression and knockdown, respectively, and successful cell modeling was verified using RT-qPCR and Western blotting. Proliferation and colony forming ability of the cells were evaluated using EdU assay and colony-forming assay, and cell migration and invasion abilities were determined using Transwell experiment. The expressions of glycolysis-related proteins M1 pyruvate kinase (PKM1) and M2 pyruvate kinase (PKM2) were detected using Western blotting. The effects of RBMX overexpression and knockdown on glycolysis in the bladder cancer cells were assessed using glucose and lactic acid detection kits. RESULTS: RT-qPCR and Western blotting confirmed successful construction of 1376 and UC-3 cell models with RBMX overexpression and knockdown. RBMX overexpression significantly inhibited the proliferation, clone formation, migration and invasion of bladder cancer cells, while RBMX knockdown produced the opposite effects. Western blotting results showed that RBMX overexpression increased the expression of PKM1 and decreased the expression of PKM2, while RBMX knockdown produced the opposite effects. Glucose consumption and lactate production levels were significantly lowered in the cells with RBMX overexpression (P < 0.05) but increased significantly following RBMX knockdown (P < 0.05). CONCLUSION: RBMX overexpression inhibits bladder cancer progression and lowers glycolysis level in bladder cancer cells by downregulating PKM2 expression, suggesting the potential of RBMX as a molecular target for diagnosis and treatment of bladder cancer.

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.

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.

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.

A novel link between melatonin and circ_0005753/PTBP1/TXNIP regulatory network in the modulation of osteogenic potential in mesenchymal stem cells.

Labeled with pluripotent potential, the transplantation of bone marrow mesenchymal stem cells (BMSCs) is considered as a promising strategy for treating osteoporosis (OP). Melatonin (MEL) has been investigated to be an essential regulator involved in bone metabolism, as well as BMSCs differentiation. Circular RNAs (circRNAs) are a unique kind of non-coding RNA and play an important regulatory role in OP. However, whether circRNAs are implicated in the effects of MEL on BMSCs osteogenic differentiation remains largely indeterminate. Expression of circ_0005753 in human BMSCs with MEL treatment, clinical specimens diagnosed with OP, either with ovariectomy (OVX)-induced mice, was measured by RT-qPCR. Western blot was conducted to analyze protein levels of osteogenesis-related molecules (Opg, RUNX2, ALP, BMP4) and TXNIP. RNA immunoprecipitation (RIP) and RNA pull-down assays were performed to validate the binding relationship among circ_0005753, PTBP1, and TXNIP. Alkaline phosphatase (ALP) and alizarin red staining (ARS) were performed to evaluate osteogenic capacity of BMSCs. OP mouse model was established by ovariectomy, as evaluated pathologic changes via hematoxylin-eosin (HE), Masson, and Immunohistochemistry (IHC) staining. Expression of circ_0005753 was remarkably decreased during MEL-induced osteogenic differentiation of BMSCs. Interestingly, not only circ_0005753 knockdown significantly promoted osteogenic differentiation of BMSCs, but circ_0005753 overexpression also weakened osteogenic differentiation induced by MEL treatment. Mechanistically, circ_0005753 maintained the stabilization of TXNIP mRNA via recruiting PTBP1. Additionally, reinforced circ_0005753 abrogated MEL-mediated protective effects on OP pathogenesis in a mouse model. This work shows that MEL facilitates osteogenic differentiation of BMSCs via the circ_0005753/PTBP1/TXNIP axis, which may shed light on the development of a novel therapeutic strategy to prevent OP.

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.

Lactate exacerbates lung damage induced by nanomicroplastic through the gut microbiota-HIF1a/PTBP1 pathway.

Exposure to nanomicroplastics (nano-MPs) can induce lung damage. The gut microbiota is a critical modulator of the gut-lung axis. However, the mechanisms underlying these interactions have not been elucidated. This study explored the role of lactate, a key metabolite of the microbiota, in the development of lung damage induced by nano-MPs (LDMP). After 28 days of exposure to nano-MPs (50-100 nm), mice mainly exhibited damage to the lungs and intestinal mucosa and dysbiosis of the gut microbiota. Lactate accumulation was observed in the lungs, intestines and serum and was strongly associated with the imbalance in lactic acid bacteria in the gut. Furthermore, no lactate accumulation was observed in germ-free mice, while the depletion of the gut microbiota using a cocktail of antibiotics produced similar results, suggesting that lactate accumulation in the lungs may have been due to changes in the gut microbiota components. Mechanistically, elevated lactate triggers activation of the HIF1a/PTBP1 pathway, exacerbating nano-MP-induced lung damage through modulation of the epithelial-mesenchymal transition (EMT). Conversely, mice with conditional knockout of Ptbp1 in the lungs (Ptbp1flfl) and PTBP1-knockout (PTBP1-KO) human bronchial epithelial (HBE) cells showed reversal of the effects of lactate through modulation of the HIF1a/PTBP1 signaling pathway. These findings indicate that lactate is a potential target for preventing and treating LDMP.

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.

A novel LINC00478 serves as a tumor suppressor in endometrial carcinoma progression.

BACKGROUND: Long noncoding RNAs (lncRNAs) are involved in the pathogenesis and progression of various cancers, but their roles in endometrial cancer (EC) are largely unknown. METHODS: The expressions of LINC00478 and PTBP1 in EC tissues were determined by RT-qPCR. Cell counting kit-8, flow cytometry and Transwell assays were executed for detecting the roles of LINC00478 in EC cells proliferation, migration and invasion. The mouse-xenograft models were established by subcutaneous injection in vivo. The interaction between LINC00478 and PTBP1 was confirmed by RNA pull-down assay and RNA-binding protein immunoprecipitation assay. RESULTS: LINC00478 was significantly down-regulated in EC tissues while compared to that in their paracancerous samples, and a higher expression level of LINC00478 was negatively correlated with clinical progress of EC patients. Functional experiments in vivo and in vitro revealed that LINC00478 overexpression could dramatically retard the proliferation of EC cells, decrease the rate of colony formation, suppress the migration and invasion abilities of EC cells in vitro and inhibit tumor growth in vivo. Mechanistically, LINC00478 regulated the expression of PTBP1, a key factor in the Warburg effect, and affected the metabolic process of EC cells. CONCLUSIONS: LINC00478 acts as a tumor suppressor in EC by negatively controlling PTBP1 expression and influencing the Warburg effect, providing a potential biomarker and therapeutic target for patients with EC.

RNA-Sequencing Analysis Indicates That N-Cadherin Promotes Prostate Cancer Progression by the Epigenetic Modification of Key Genes.

N-cadherin (cadherin-2 [CDH2]) is widely known as the promoter of prostate cancer (PCa) invasion and castration resistance. However, the biological mechanism of N-cadherin in PCa progression is unclear. In this study, we overexpressed N-cadherin in LNCaP cells and downregulated N-cadherin in PC3 cells by lentiviral transduction. Then, differentially expressed genes (DEGs) and dysregulated biological functions were investigated through RNA sequencing (RNA-seq) analyses. We found 13 long noncoding RNA (lncRNA) transcripts, 72 messenger RNA (mRNA) transcripts, and 3 integrated genes were dysregulated by N-cadherin. In the disease enrichment, bone cancer, and neurodegenerative and nervous system diseases were associated with N-cadherin in the circular RNA (circRNA; PC3 versus [vs.,/] LNCaP [PC3/LNCaP] comparison) and DEG analysis (LNCaP_oe_CDH2 vs. LNCaP_oe_NC [LNCaP_oe_CDH2/NC] comparison). Epigenetic reprogramming, such as nucleic acid binding, and chromatin and histone modifications, was enriched in Gene Ontology (GO) analysis (DEGs in LNCaP_oe_CDH2/NC and PC3_sh_NC/CDH2, and host genes of circRNA in PC3/LNCaP). Transcriptional misregulation in cancer, post-translational protein modification, gene expression, and generic transcription pathways were dysregulated in the pathway enrichment analysis (host genes of circRNA in PC3/LNCaP, and DEGs in LNCaP_oe_CDH2/NC and PC3_sh_NC/CDH2). Verifying DEGs through TCGA-PRAD dataset revealed six oncogenes (ARHGEF1, GRAMD1A, GTF2H4, MAPK8IP3, POLD1, and PTBP1) that were commonly upregulated by N-cadherin and in advanced PCa stages. In summary, we identified several oncogenes and biological functions associated with N-cadherin expression in PCa cells. N-cadherin may trigger epigenetic reprogramming in PCa cells to promote tumor progression.

Dynamics of TERT regulation via alternative splicing in stem cells and cancer cells.

Part of the regulation of telomerase activity includes the alternative splicing (AS) of the catalytic subunit telomerase reverse transcriptase (TERT). Although a therapeutic window for telomerase/TERT inhibition exists between cancer cells and somatic cells, stem cells express TERT and rely on telomerase activity for physiological replacement of cells. Therefore, identifying differences in TERT regulation between stem cells and cancer cells is essential for developing telomerase inhibition-based cancer therapies that reduce damage to stem cells. In this study, we measured TERT splice variant expression and telomerase activity in induced pluripotent stem cells (iPSCs), neural progenitor cells (NPCs), and non-small cell lung cancer cells (NSCLC, Calu-6 cells). We observed that a NOVA1-PTBP1-PTBP2 axis regulates TERT alternative splicing (AS) in iPSCs and their differentiation into NPCs. We also found that splice-switching of TERT, which regulates telomerase activity, is induced by different cell densities in stem cells but not cancer cells. Lastly, we identified cell type-specific splicing factors that regulate TERT AS. Overall, our findings represent an important step forward in understanding the regulation of TERT AS in stem cells and cancer cells.

PTBP1 regulates injury responses and sensory pathways in adult peripheral neurons.

Polypyrimidine tract binding protein 1 (PTBP1) is thought to be expressed only at embryonic stages in central neurons. Its down-regulation triggers neuronal differentiation in precursor and non-neuronal cells, an approach recently tested for generation of neurons de novo for amelioration of neurodegenerative disorders. Moreover, PTBP1 is replaced by its paralog PTBP2 in mature central neurons. Unexpectedly, we found that both proteins are coexpressed in adult sensory and motor neurons, with PTBP2 restricted mainly to the nucleus, while PTBP1 also shows axonal localization. Levels of axonal PTBP1 increased markedly after peripheral nerve injury, and it associates in axons with mRNAs involved in injury responses and nerve regeneration, including importin ß1 (KPNB1) and RHOA. Perturbation of PTBP1 affects local translation in axons, nociceptor neuron regeneration and both thermal and mechanical sensation. Thus, PTBP1 has functional roles in adult axons. Hence, caution is required before considering targeting of PTBP1 for therapeutic purposes.

NAT10-mediated RNA acetylation enhances HNRNPUL1 mRNA stability to contribute cervical cancer progression.

N4-acetylcytidine (ac4C) is a lately discovered nucleotide modification that has been shown to be closely implicated in cancer. N-acetyltransferase10(NAT10) acts as an enzyme that regulates mRNA acetylation modifications. Currently, the role of NAT10-mediated RNA acetylation modification in cervical cancer remains to be elucidated. On the basis of transcriptome analysis of TCGA and GEO open datasets (GSE52904, GSE29570, GSE122697), NAT10 is upregulated in cervical cancer tissues and correlated with poor prognosis. Knockdown of NAT10 suppressed the cell proliferation, invasion, and migration of cervical cancer cells. The in vivo oncogenic function of NAT10 was also confirmed in xenograft models. Combined RNA-seq and acRIP-seq analysis revealed HNRNPUL1 as the target of NAT10 in cervical cancer. NAT10 positively regulate HNRNPUL1 expression by promoting ac4C modification and stability of HNRNPUL1 mRNA. Furthermore, depletion of HNRNPUL1 suppressed the cell division, invasion, and migration of cervical cancer. HNRNPUL1 overexpression partially restored cellular function in cervical cancer cells with NAT10 knockdown. Thus, this study demonstrates that NAT10 contributes to cervical cancer progression by enhancing HNRNPUL1 mRNA stability via ac4C modification, and NAT10-ac4C-HNRNPUL1 axis might be a potential target for cervical cancer therapy.