LINC01432 binds to CELF2 in newly diagnosed multiple myeloma promoting short progression-free survival to standard therapy.

Multiple Myeloma (MM) is a highly prevalent and incurable form of cancer that arises from malignant plasma cells, with over 35,000 new cases diagnosed annually in the United States. While there are a growing number of approved therapies, MM remains incurable and nearly all patients will relapse and exhaust all available treatment options. Mechanisms for disease progression are unclear and in particular, little is known regarding the role of long non-coding RNAs (lncRNA) in mediating disease progression and response to treatment. In this study, we used transcriptome sequencing to compare newly diagnosed MM patients who had short progression-free survival (PFS) to standard first-line treatment (PFS < 24 months) to patients who had prolonged PFS (PFS > 24 months). We identified 157 differentially upregulated lncRNAs with short PFS and focused our efforts on characterizing the most upregulated lncRNA, LINC01432. We investigated LINC01432 overexpression and CRISPR/Cas9 knockdown in MM cell lines to show that LINC01432 overexpression significantly increases cell viability and reduces apoptosis, while knockdown significantly reduces viability and increases apoptosis, supporting the clinical relevance of this lncRNA. Next, we used individual-nucleotide resolution cross-linking immunoprecipitation with RT-qPCR to show that LINC01432 directly interacts with the RNA binding protein, CELF2. Lastly, we showed that LINC01432-targeted locked nucleic acid antisense oligonucleotides reduce viability and increases apoptosis. In summary, this fundamental study identified lncRNAs associated with short PFS to standard NDMM treatment and further characterized LINC01432, which inhibits apoptosis.

Loss of CELF2 promotes skin tumorigenesis and increases drug resistance.

BACKGROUND: CELF2 belongs to the CELF RNA-binding protein family and exhibits antitumor activity in various tumor models. Analysis of the pan-cancer TCGA database reveals that CELF2 expression strongly correlates with favorable prognosis among cancer patients. The function of CELF2 in nonmelanoma skin cancer has not been studied. METHODS: We used shRNA-mediated knockdown (KD) of CELF2 expression in human squamous cell carcinoma (SCC) cells to investigate how CELF2 impacted SCC cell proliferation, survival, and xenograft tumor growth. We determined CELF2 expression in human SCC tissues and adjacent normal skin using immunofluorescence staining. Additionally, we investigated the changes in CELF2 and its target gene expression during UV-induced and chemical-induced skin tumorigenesis by western blotting. RESULTS: CELF2 KD significantly increased SCC cell proliferation, colony growth, and SCC xenograft tumor growth in immunodeficient mice. CELF2 KD in SCC cells led to activation of KRT80 and GDF15, which can potentially promote cell proliferation and tumor growth. While control SCC cells were sensitive to anticancer drugs such as doxorubicin, SCC cells with CELF2 KD became resistant to drug-induced tumor growth retardation. Finally, we found CELF2 expression diminished during both UV- and chemical-induced skin tumorigenesis in mice, consistent with reduced CELF2 expression in human SCC tumors compared to adjacent normal skin. CONCLUSION: This study shows for the first time that CELF2 loss occurs during skin tumorigenesis and increases drug resistance in SCC cells, highlighting the possibility of targeting CELF2-regulated pathways in skin cancer prevention and therapies.

CELF2 Deficiency Demonstrates Autism-Like Behaviors and Interferes with Late Development of Cortical Neurons in Mice.

CELF2 variants have been linked to neurodevelopmental disorders (NDD), including autism spectrum disorder (ASD). However, the molecular mechanisms remain unclear. We generated Celf2 Nestin-Cre knockout mice.Our findings revealed that Celf2 Nestin-Cre heterozygous knockout mice exhibited social impairment and anxiety, an autism-like behavior, though no manifestations of repetitive stereotyped behavior, learning cognitive impairment, or depression were observed. Immunofluorescence assay showed an underdeveloped cerebral cortex with significantly reduced cortical thickness, albeit without abnormal cell density. Further in vitro neuronal culture demonstrated a significant reduction in dendritic spine density and affected synaptic maturation in Celf2 deficient mice, with no notable abnormalities in total neurite and axon length. RNA-seq and RIP-seq analysis of the cerebral cortex revealed differentially expressed genes post Celf2 gene knockout compared with the control group. Enrichment analysis highlighted significant enrichment in dendrite and synapse-related biological processes and pathways. Our study delineated the behavioral and neurodevelopmental phenotypes of Celf2, suggesting its potential involvement in autism through the regulation of target genes associated with dendritic spines and synapse development. Further research is needed to elucidate the specific mechanisms involved.

The role of CELF family in neurodevelopment and neurodevelopmental disorders.

RNA-binding proteins (RBPs) bind to RNAs and are crucial for regulating RNA splicing, stability, translation, and transport. Among these proteins, the CUGBP Elav-like family (CELF) is a highly conserved group crucial for posttranscriptional regulation by binding to CUG repeats. Comprising CELF1-6, this family exhibits diverse expression patterns and functions. Dysregulation of CELF has been implicated in various neural disorders, encompassing both neurodegenerative and neurodevelopmental conditions, such as Alzheimer's disease and autism. This article aims to provide a comprehensive summary of the CELF family's role in neurodevelopment and neurodevelopmental disorders. Understanding CELF's mechanisms may offer clues for potential therapeutic strategies by regulating their targets in neurodevelopmental disorders.

Downregulated antisense lncRNA ENTPD3-AS1 contributes to the development of lung adenocarcinoma.

The poor outcome of patients with lung adenocarcinoma (LUAD) highlights the importance to identify novel effective prognostic markers and therapeutic targets. Long noncoding RNAs (lncRNAs) have generally been considered to serve important roles in tumorigenesis and the development of various types of cancer, including LUAD. Here, we aimed to investigate the role of ENTPD3-AS1 (ENTPD3 Antisense RNA 1) in LUAD and to explore its potential mechanisms by performing comprehensive bioinformatic analyses. The regulatory effect of ENTPD3-AS1 on the expression of NR3C1 was validated by siRNA-based silencing. The effect of miR-421 on the modulation of NR3C1 was determined by miRNA mimics and inhibitors transfection. ENTPD3-AS1 was expressed at lower levels in tumor parts and negatively correlated with unfavorable prognosis in LUAD patients. It exerted functions as a tumor suppressor gene by competitively binding to oncomir, miR-421, thereby attenuating NR3C1 expression. Transfection of lung cancer A549 cells with miR-421 mimics decreased the expression of NR3C1. Transfection of lung cancer A549 cells with miR-421 inhibitors increased the expression of NR3C1 with lower cellular functions as proliferation and migration via epithelial-mesenchymal transition. In addition, inhibition of ENTPD3-AS1 by siRNA transfection decreased the levels of NR3C1, supporting the ENTPD3-AS1/miR-421/NR3C1 cascade. Moreover, the bioinformatic analysis also showed that ENTPD3-AS1 could interact with the RNA-binding proteins (RBPs), CELF2 and QKI, consequently regulating RNA expression and processing. Taken together, we identified that ENTPD3-AS1 and its indirect target NR3C1 can act as novel biomarkers for determining the prognosis of patients with LUAD, and further study is required.

CELF2 Sustains a Proliferating/OLIG2+ Glioblastoma Cell Phenotype via the Epigenetic Repression of SOX3.

Glioblastomas (GBs) are incurable brain tumors. The persistence of aggressive stem-like tumor cells after cytotoxic treatments compromises therapeutic efficacy, leading to GBM recurrence. Forcing the GBM cells to irreversibly abandon their aggressive stem-like phenotype may offer an alternative to conventional cytotoxic treatments. Here, we show that the RNA binding protein CELF2 is strongly expressed in mitotic and OLIG2-positive GBM cells, while it is downregulated in differentiated and non-mitotic cells by miR-199a-3p, exemplifying GBM intra-tumor heterogeneity. Using patient-derived cells and human GBM samples, we demonstrate that CELF2 plays a key role in maintaining the proliferative/OLIG2 cell phenotype with clonal and tumorigenic properties. Indeed, we show that CELF2 deficiency in patient-derived GSCs drastically reduced tumor growth in the brains of nude mice. We further show that CELF2 promotes TRIM28 and G9a expression, which drive a H3K9me3 epigenetic profile responsible for the silencing of the SOX3 gene. Thus, CELF2, which is positively correlated with OLIG2 and Ki67 expression in human GBM samples, is inversely correlated with SOX3 and miR-199a-3p. Accordingly, the invalidation of SOX3 in CELF2-deficient patient-derived cells rescued proliferation and OLIG2 expression. Finally, patients expressing SOX3 above the median level of expression tend to have a longer life expectancy. CELF2 is therefore a crucial target for the malignant potential of GBM and warrants attention when developing novel anticancer strategies.

CircLIFR Inhibits Non-small Cell Lung Cancer Progression by Acting as a miR-429 Sponge to Enhance CELF2 Expression.

Lung cancer is the most commonly diagnosed cancer and the leading reason for tumor-related mortality, while non-small cell lung cancer (NSCLC) is the most usual type of lung cancer. Circular RNAs (circRNAs) have emerged as vital regulators in the development of human cancers, including NSCLC. We aimed to explore the functions of circRNA leukemia inhibitory factor receptor (circLIFR) in NSCLC progression. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to quantify the expression of circLIFR, microRNA-429 (miR-429), and Elav-like family member 2 (CELF2) in NSCLC tissues and cells. Cell proliferation capability of NSCLC cells was determined by Cell Counting Kit-8 (CCK-8) and colony formation assays. The flow cytometry assay was performed to evaluate cell-cycle distribution and apoptosis of NSCLC cells. The abilities of migration and invasion were measured by transwell assay. In addition, the activities of caspase 3 and caspase 9 were measured by the assay kits. The interaction relationship between miR-429 and circLIFR or CELF2 was analyzed by dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays. The expression levels of related proteins were examined by Western Blot assay. The xenograft experiment was established to explore the role of circLIFR in vivo. CircLIFR, circular, and stable transcript in NSCLC cells, was decreased more than 2 folds in NSCLC tissues and cells than controls (P < 0.0001). Importantly, overexpression of circLIFR impeded cell proliferation, migration, invasion, and inactivated protein kinase B (AKT)/phosphatase and tensin homolog (PTEN)-signaling pathways while enhanced apoptosis and cell-cycle arrest in NSCLC cells, which was overturned by upregulation of miR-429 or silencing of CELF2. Furthermore, the upregulation of circLIFR inhibited NSCLC tumor growth in vivo. Overexpression of circLIFR could suppress NSCLC progress by acting as a sponge of miR-429 to regulate the expression of CELF2 and PTEN/AKT-signaling pathways in NSCLC.

ELK1-Induced upregulation of long non-coding TNK2-AS1 promotes the progression of acute myeloid leukemia by EZH2-mediated epigenetic silencing of CELF2.

Acute myeloid leukemia (AML) is the second most common hematological malignancy after lymphoma in the world. Long non-coding RNAs (LncRNAs) have been suggested as key regulators of cancer development and progression in AML. As a member of lncRNA family, the biological role and mechanisms of tyrosine kinase non receptor 2 antisense RNA 1 (TNK2-AS1) in AML is still unclear. The expression of TNK2-AS1 was measured with RT-qPCR in AML cell lines. The changes of the proliferation, apoptosis, and differentiation in TNK2-AS1 shRNA-transfected HL-60 and THP-1 cells were detected with CCK-8, EdU, flow cytometry, Western blot, and NBT assays. Molecular control of TNK2-AS1 on CUGBP Elav-like family member 2 (CELF2) and ETS domain-containing protein-1 (ELK1) on TNK2-AS1 was assessed by chromatin immunoprecipitation (ChIP), RT-qPCR, Western blot, and RNA immunoprecipitation (RIP) assays. TNK2-AS1 expression was upregulated in AML cell lines and negatively correlated with survival patients. Knockdown of TNK2-AS1 markedly reduced AML cell proliferation and promoted apoptosis and differentiation. Likewise, TNK2-AS1 knockdown significantly suppressed tumor growth in vivo. Mechanistically, the upregulation of TNK2-AS1 was activated by transcription factor ELK1. We also uncovered that TNK2-AS1 exerted tumor-promoting effect through silencing CELF2 via binding with EZH2, thus activating PI3K/Akt pathway in AML cells. Elevated expression of TNK2-AS1 was induced by ELK1 and facilitated AML progression by suppressing CELF2 expression via EZH2-mediated epigenetic silencing, suggesting TNK2-AS1 may be a promising therapeutic target and prognostic marker for AML patients.

The Histone Demethylase HR Suppresses Breast Cancer Development through Enhanced CELF2 Tumor Suppressor Activity.

The hairless (HR) gene encodes a transcription factor with histone demethylase activity that is essential for development and tissue homeostasis. Previous studies suggest that mutational inactivation of HR promotes tumorigenesis. To investigate HR mutations in breast cancer, we performed targeted next-generation sequencing using DNA isolated from primary breast cancer tissues. We identified HR somatic mutations in approximately 15% of the patient cohort (n = 85), compared with 23% for BRCA2, 13% for GATA3, 7% for BRCA1, and 3% for PTEN in the same patient cohort. We also found an average 23% HR copy number loss in breast cancers. In support of HR's antitumor functions, HR reconstitution in HR-deficient human breast cancer cells significantly suppressed tumor growth in orthotopic xenograft mouse models. We further demonstrated that HR's antitumor activity was at least partly mediated by transcriptional activation of CELF2, a tumor suppressor with RNA-binding activity. Consistent with HR's histone demethylase activity, pharmacologic inhibition of histone methylation suppressed HR-deficient breast cancer cell proliferation, migration and tumor growth. Taken together, we identified HR as a novel tumor suppressor that is frequently mutated in breast cancer. We also showed that pharmacologic inhibition of histone methylation is effective in suppressing HR-deficient breast tumor growth and progression.

N6-methyladenosine-mediated CELF2 regulates CD44 alternative splicing affecting tumorigenesis via ERAD pathway in pancreatic cancer.

BACKGROUND: Alternative splicing (AS) of genes has been found to affect gene stability, and its abnormal regulation can lead to tumorigenesis. CELF2 is a vital splicing factor to participate in mRNA alternative splicing. Its downregulation has been confirmed to promote the occurrence and development of pancreatic cancer (PC). However, the regulatory role and mechanisms in PC has not been elucidated. RESULTS: CELF2 was downregulated in PC tissues, which affected tumor TNM stage and tumor size, and low expression of CELF2 indicated a poor prognosis of PC. In vivo and in vitro experiments showed that abnormal expression of CELF2 affected the stemness, apoptosis, and proliferation of PC cells. Furthmore, we also found that CELF2 was targeted by ALKBH5 for m6A modification, leading to CELF2 degradation by YTHDF2. Bioinformatic analysis of AS model based on the TCGA database indicated that CELF2 could target CD44 to form different spliceosomes, thereby affecting the biological behavior of PC cells. The conversion of CD44s to CD44V is the key to tumorigenesis. Transcriptomic analysis was conducted to reveal the mechanism of CELF2-mediated CD44 AS in PC. We found that CELF2-mediated splicing of CD44 led to changes in the level of endoplasmic reticulum stress, further regulating the endoplasmic reticulum-associated degradation (ERAD) signaling pathway, thereby affecting apoptosis and cell stemness. In addition, ERAD signaling pathway inhibitor, EerI, could effectively reverse the effect of CD44 on tumors. CONCLUSIONS: This study indicates that N6-methyladenosine-mediated CELF2 promotes AS of CD44, affecting the ERAD pathway and regulating the biological behavior of PC cells. CELF2 is expected to be a new target for targeted-drug development.

MiR-210-3p targets CELF2 to facilitate progression of lung squamous carcinoma through PI3K/AKT pathway.

This study examined the internal mechanism of miR-210-3p/CELF2 in LUSC. Expression data of mRNAs and miRNAs in LUSC were acquired from TCGA and subjected to differential expression analysis. qRT-PCR was applied to examine miR-210-3p and CELF2 expression. Besides, western blot was utilized to evaluate protein expression of CELF2 and PI3K/AKT pathway-related proteins. Dual-luciferase reporter analysis was conducted to validate targeting relationship between miR-210-3p and CELF2. Additionally, CCK-8, colony formation, transwell and flow cytometry were employed to respectively test proliferation, migration, invasion abilities and cell cycle distribution. Xenograft tumor models were used to evaluate the influence of miR-210-3p and CELF2 on tumor growth. MiR-210-3p was highly expressed, while CELF2 was less expressed in LUSC cells. Besides, miR-210-3p could downregulate CELF2 expression. Cell functional assay verified that miR-210-3p accelerated aggressive behaviors of LUSC cells. Additionally, rescue assay suggested that miR-210-3p downregulated CELF2 level to stimulate LUSC cell phenotypes and cell cycle progression through PI3K/AKT pathway. Moreover, miR-210-3p/CELF2 stimulated the tumor growth in vivo. To sum up, miR-210-3p modulated CELF2 expression, thus affecting cell phenotypes and cell cycle distribution in LUSC through PI3K/AKT pathway.

Downregulation of STAT3 in Epstein-Barr Virus-Positive Hodgkin Lymphoma.

STAT3 is a transcription factor which is activated via various signaling transduction pathways or Epstein-Barr virus (EBV) infection and plays an oncogenic role in lymphoid malignancies including Hodgkin lymphoma (HL). The tumor cells of HL are derived from germinal center B-cells and transformed by chromosomal rearrangements, aberrant signal transduction, deregulation of developmental transcription factors, and EBV activity. HL cell lines represent useful models to investigate molecular principles and deduced treatment options of this malignancy. Using cell line L-540, we have recently shown that constitutively activated STAT3 drives aberrant expression of hematopoietic NKL homeobox gene HLX. Here, we analyzed HL cell line AM-HLH which is EBV-positive but, nevertheless, HLX-negative. Consistently, AM-HLH expressed decreased levels of STAT3 proteins which were additionally inactivated and located in the cytoplasm. Combined genomic and expression profiling data revealed several amplified and overexpressed gene candidates involved in opposed regulation of STAT3 and EBV. Corresponding knockdown studies demonstrated that IRF4 and NFATC2 inhibited STAT3 expression. MIR155 (activated by STAT3) and SPIB (repressed by HLX) showed reduced and elevated expression levels in AM-HLH, respectively. However, treatment with IL6 or IL27 activated STAT3, elevated expression of HLX and MIR155, and inhibited IRF4. Taken together, this cell line deals with two conflicting oncogenic drivers, namely, JAK2-STAT3 signaling and EBV infection, but is sensitive to switch after cytokine stimulation. Thus, AM-HLH represents a unique cell line model to study the pathogenic roles of STAT3 and EBV and their therapeutic implications in HL.

miR-363-3p induces EMT via the Wnt/ß-catenin pathway in glioma cells by targeting CELF2.

In our previous study, we reported that CELF2 has a tumour-suppressive function in glioma. Here, we performed additional experiments to elucidate better its role in cancer. The expression profile of CELF2 was analysed by the GEPIA database, and Kaplan-Meier curves were used to evaluate the overall survival rates. Four different online databases were used to predict miRNAs targeting CELF2, and the luciferase assay was performed to identify the binding site. The biological effects of miR-363-3p and CELF2 were also investigated in vitro using MTT, Transwell, and flow cytometry assays. Western blotting, qPCR, and TOP/FOP flash dual-luciferase assays were performed to investigate the impact of miR-363-3p and CELF2 on epithelial-to-mesenchymal transition (EMT) and the Wnt/ß-catenin pathway. The effect of miR-363-3p was also tested in vivo using a xenograft mouse model. We observed an abnormal expression pattern of CELF2 in glioma cells, and higher CELF2 expression correlated with better prognosis. We identified miR-363-3p as an upstream regulator of CELF2 and confirmed its direct binding to the 3'-untranslated region of CELF2. Cell function experiments showed that miR-363-3p affected multiple aspects of glioma cells. Suppressing miR-363-3p expression inhibited glioma cell proliferation and invasion, as well as promoted cell death via attenuating EMT and blocking the Wnt/ß-catenin pathway. These effects could be abolished by the downregulation of CELF2. Treatment with ASO-miR-363-3p decreased tumour size and weight in nude mice. In conclusion, miR-363-3p induced the EMT, which resulted in increased migration and invasion and reduced apoptosis in glioma cell lines, via the Wnt/ß-catenin pathway by targeting CELF2.

CircPTK2 inhibits the tumorigenesis and metastasis of gastric cancer by sponging miR-134-5p and activating CELF2/PTEN signaling.

BACKGROUND: CircRNAs are a new subset of noncoding RNAs formed by covalent closed loops and play crucial roles in the regulation of cancer gene expression. However, the roles and underlying mechanisms of circRNAs in gastric cancer (GC) remain indistinct. This study aimed to explore the role and mechanism of hsa_circ_0006421 (circPTK2) in GC. METHODS: The differential expression of circRNAs between GC tissues and adjacent normal tissues were identified by a circRNA expression profiling. Associations of circPTK2 or miR-134-5p expression with clinicopathological characteristics and prognosis of GC patients were analyzed by chi-square of Fisher's exact tests and Kaplan-Meier analysis. CCK8, colony formation, EdU assays and animal models were performed to assess the effects of circPTK2 on proliferation and invasion of GC cells. CircPTK2-specific probes were used to purify the RNA pulled down from the circPTK2, and enrichment of circPTK2 and miR-134-5p was detected by qRT-PCR. The effects of circPTK2 on miR-134-5p expression and CELF2/PTEN signaling were examined by qRT-PCR and Western blotting analysis. RESULTS: Low expression of circPTK2 and high expression of miR-134-5p were related to the poor survival, and high expression of miR-134-5p was related to the tumor recurrence in GC patients. Overexpressing circPTK2 suppressed the proliferation, colony formation, DNA synthesis and cell invasion as well as xenograft tumor growth and lung metastasis in vitro and in vivo, whereas silencing circPTK2 had the opposite effects. Moreover, circPTK2 was negatively correlated and co-localized with miR-134-5p in the cytoplasm of GC tissue cells. circPTK2 bound to and sponged miR-134-5p in GC cells, and miR-134-5p facilitated cell growth and invasion but attenuated circPTK2 induced tumor suppressive effects and CELF2/PTEN signaling activation in GC cells. CONCLUSIONS: circPTK2 functions as a tumor suppressor in GC by sponging miR-134-5p and activating the CELF2/PTEN axis.

LncRNA RHPN1-AS1 promotes the progression of nasopharyngeal carcinoma by targeting CELF2 expression.

This study aims to investigate the role of lncRNA RHPN1-AS1 in NPC and its potential regulatory mechanism. The expression of RHPN1-AS1 in tissues and cells was measured by qRT-PCR. The effect of RHPN1-AS1 silencing on biological functions of NPC cells was detected by CCK-8, colony formation, flow cytometry, wound healing, and transwell assays. The protein expression was measured by western blot. The RBPs of RHPN1-AS1 were predicted by Starbase and LncTar, and verify by RIP assay. ESTIMATE was used to analyze the relationship between CELF2 expression and tumor purity. GSEA was used to analyze the downstream signaling pathway of CELF2. In our study, RHPN1-AS1 was up-regulated in NPC tissues and cells. RHPN1-AS1 silencing inhibited cell viability, capacity of proliferation, migration and invasion, promoted apoptosis, decreased protein expression of Bcl-2, MMP2/9, increased protein expression of Bax, caspase-3, and TIMP2 of NPC cells. CELF2 was a target of RHPN1-AS1 and was down-regulated in NPC tissues and cells. CELF2 level was associated with tumor purity negatively. Low expression of CELF2 activated mTORC1 signaling pathway and increased protein expression of p-mTORC1/mTORC1 and p-P70S6K/P70S6K. RHPN1-AS1 silencing eliminated the activating effect of CELF2 silencing on mTORC1 signaling pathway. Moreover, CELF2 silencing reversed the inhibitory effect of RHPN1-AS1 on NPC progression. In conclusion, our findings indicated that RHPN1-AS1 plays an important role in NPC via activating mTORC1 signaling which is modulated by CELF2. RHPN1-AS1 may serve as a potential therapeutic target for NPC treatment.

CELF2 is a candidate prognostic and immunotherapy biomarker in triple-negative breast cancer and lung squamous cell carcinoma: A pan-cancer analysis.

CUGBP Elav-like family member 2(CELF2) plays crucial roles in the development and activation of T cell. However, the impacts of CELF2 on tumour-infiltrating immune cells (TIICs) and clinical outcomes of tumours remain unclear. In this study, we found that elevated CELF2 expression was markedly correlated with prolonged survival in multiple tumours, particularly in breast and lung cancers. Notably, CELF2 only impacted the prognosis of triple-negative breast cancer (TNBC) with lymph node metastasis. Further investigation showed CELF2 expression was positively correlated with the infiltration abundance of dendritic cells (DCs), CD8+ T cells and neutrophils in breast invasive carcinoma (BRCA) and DCs in lung squamous cell carcinoma (LUSC). CELF2 also had strong correlations with markers of diverse TIICs such as T cells, tumour-associated macrophages and DCs in BRCA and LUSC. Importantly, CELF2 was significantly associated with plenty of immune checkpoint molecules (ICMs) and outperformed five prevalent biomarkers including PD-1, PD-L1, CTLA-4, CD8 and tumour mutation burden in predicting immunotherapeutic responses. Immunohistochemistry also revealed lower protein levels of CELF2 in TNBC and LUSC compared to normal tissues, and patients with high expression showed significantly prolonged prognosis. In conclusion, we demonstrated that increased CELF2 expression was closely related to better prognosis and superior TIIC infiltration and ICM expression, particularly in BRCA and LUSC. CELF2 also performed well in evaluating the immunotherapeutic efficacy, suggesting CELF2 might be a promising biomarker.

Nucleocytoplasmic transport of the RNA-binding protein CELF2 regulates neural stem cell fates.

The development of the cerebral cortex requires balanced expansion and differentiation of neural stem/progenitor cells (NPCs), which rely on precise regulation of gene expression. Because NPCs often exhibit transcriptional priming of cell-fate-determination genes, the ultimate output of these genes for fate decisions must be carefully controlled in a timely fashion at the post-transcriptional level, but how that is achieved is poorly understood. Here, we report that de novo missense variants in an RNA-binding protein CELF2 cause human cortical malformations and perturb NPC fate decisions in mice by disrupting CELF2 nucleocytoplasmic transport. In self-renewing NPCs, CELF2 resides in the cytoplasm, where it represses mRNAs encoding cell fate regulators and neurodevelopmental disorder-related factors. The translocation of CELF2 into the nucleus releases mRNA for translation and thereby triggers NPC differentiation. Our results reveal that CELF2 translocation between subcellular compartments orchestrates mRNA at the translational level to instruct cell fates in cortical development.

Hypoxic colorectal cancer-secreted exosomes deliver miR-210-3p to normoxic tumor cells to elicit a protumoral effect.

Hypoxia, the most common feature in the tumor microenvironment, is closely related to tumor malignant progression and poor patient's prognosis. Exosomes, initially recognized as cellular "garbage dumpsters", are now known to be important mediums for mediating cellular communication in tumor microenvironment. However, the mechanisms of hypoxic tumor cell-derived exosomes facilitate colorectal cancer progression still need further exploration. In the present study, we found that exosomes from hypoxic colorectal cancer cells (H-Exos) promoted G1-S cycle transition and proliferation while preventing the apoptosis of colorectal cancer cells by transmitting miR-210-3p to normoxic tumor cells. Mechanistic investigation indicated that miR-210-3p from H-Exos elicited its protumoral effect via suppressing CELF2 expression. A preclinical study further confirmed that H-Exos could promote tumorigenesis in vivo. Clinically, the expression of miR-210-3p in circulating plasma exosomes was markedly upregulated in colorectal cancer patients, which were closely associated with multiple unfavorable clinicopathological features. Taken together, these results suggest that hypoxia may stimulate colorectal cancer cells to secrete miR-210-3p-enriched exosomes in tumor microenvironment, which elicit protumoral effects by inhibiting CELF2 expression. These findings provide new insights on the mechanism of colorectal cancer progression and potential therapeutic targets for colorectal cancer.

Triclosan regulates alternative splicing events of nerve-related genes through RNA-binding protein CELF2 to induce zebrafish neurotoxicity.

Herein, we demonstrated that triclosan (TCS) induced neurotoxicity mediated by pre-mRNA alternative splicing (AS). TCS exposure resulted in a series of phenotypic malformations, abnormal locomotor behavior, circadian rhythm disorder and inhibited AChE activity. High throughput mRNA sequencing revealed that TCS regulated the AS events of nerve-related genes. Meanwhile, abnormal expression was observed in marker genes related to nerve cell migration, axon guidance and myelination. The expression of mitochondrial apoptosis activator bcl2l11 was significantly increased under TCS exposure. Interestingly, CELF2 as one of the important RNA-binding proteins was closely related to the AS events, and its mRNA and protein expression levels were significantly increased in zebrafish brain under acute or chronic TCS exposure. Functional knock-down and over-expression of celf2 confirmed that TCS led to nervous system injury and developmental defects through the CELF2-mediated AS events of genes (mbpa, mef2d, u2af2b and matn3b). Histopathological injury, phenotypic malformation, abnormal locomotor behavior and changes in neuromarkers all confirmed the biological functions of CELF2 in zebrafish brain. These findings demonstrate that TCS might regulate some of the AS events of nerve-related genes through upregulating the expression of CELF2. Thus, CELF2 may serve as a target for the prevention, diagnosis and treatment of contaminant-induced neurological diseases.

Activation of MAP3K DLK and LZK in Purkinje cells causes rapid and slow degeneration depending on signaling strength.

The conserved MAP3K Dual-Leucine-Zipper Kinase (DLK) and Leucine-Zipper-bearing Kinase (LZK) can activate JNK via MKK4 or MKK7. These two MAP3Ks share similar biochemical activities and undergo auto-activation upon increased expression. Depending on cell-type and nature of insults DLK and LZK can induce pro-regenerative, pro-apoptotic or pro-degenerative responses, although the mechanistic basis of their action is not well understood. Here, we investigated these two MAP3Ks in cerebellar Purkinje cells using loss- and gain-of function mouse models. While loss of each or both kinases does not cause discernible defects in Purkinje cells, activating DLK causes rapid death and activating LZK leads to slow degeneration. Each kinase induces JNK activation and caspase-mediated apoptosis independent of each other. Significantly, deleting CELF2, which regulates alternative splicing of Map2k7, strongly attenuates Purkinje cell degeneration induced by LZK, but not DLK. Thus, controlling the activity levels of DLK and LZK is critical for neuronal survival and health.