Generation and characterization of a human iPSC line and gene-corrected isogenic line derived from a patient with a CELF2 gene mutation.

The identification of neurodevelopmental defects in a patient harboring a heterozygous de novo missense variant (NM_006561.4, c.1517G > A, p.Arg506His) within the CELF2 gene. Here, we describe the establishment of a patient-derived induced pluripotent stem cell (iPSC) line, alongside an isogenic gene-corrected iPSC line, achieved through CRISPR/Cas9 genome editing. These lines exhibit the expression of pluripotency markers, demonstrate differentiation potential into all three germ layers, and maintain a normal karyotype. These iPSC lines serve as valuable tools for investigating the consequences of CELF2 related neurodevelopmental disorders.

Comprehensive analysis of lncRNA-miRNA-mRNA ceRNA network and key genes in granulosa cells of patients with biochemical primary ovarian insufficiency.

Primary ovarian insufficiency (POI) is a common condition leading to the pathological decline of ovarian function in women of reproductive age, resulting in amenorrhea, hypogonadism, and infertility. Biochemical premature ovarian insufficiency (bPOI) is an intermediate stage in the pathogenesis of POI in which the fertility of patients has been reduced. Previous studies suggest that granulosa cells (GCs) play an essential role in the pathogenesis of POI, but their pathogenetic mechanisms remain unclear. To further explore the potential pathophysiological mechanisms of GCs in POI, we constructed a molecular long non-coding RNA (lncRNA)-microRNA (miRNA)-messenger RNA (mRNA) network using GC expression data collected from biochemical premature ovarian failure (bPOI) patients in the GEO database. We discovered that the GCs of bPOI patients had differential expression of 131 mRNAs, 191 lncRNAs, and 28 miRNAs. By systematic network analysis, we identified six key genes, including SRSF1, PDIA5, NEURL1B, UNK, CELF2, and CFL2, and five hub miRNAs, namely hsa-miR-27a-3p, hsa-miR-24-3p, hsa-miR-22-3p, hsa-miR-129-5p, and hsa-miR-17-5p, and the results suggest that the expression of these key genes may be regulated by two hub miRNAs, hsa-miR-27a-3p and hsa-miR-17-5p. Additionally, a POI model in vitro was created to confirm the expression of a few important genes. In this study, we discovered a unique lncRNA-miRNA-mRNA network based on the ceRNA mechanism in bPOI for the first time, and we screened important associated molecules, providing a partial theoretical foundation to better understand the pathogenesis of POI.

Celf4 controls mRNA translation underlying synaptic development in the prenatal mammalian neocortex.

Abnormalities in neocortical and synaptic development are linked to neurodevelopmental disorders. However, the molecular and cellular mechanisms governing initial synapse formation in the prenatal neocortex remain poorly understood. Using polysome profiling coupled with snRNAseq on human cortical samples at various fetal phases, we identify human mRNAs, including those encoding synaptic proteins, with finely controlled translation in distinct cell populations of developing frontal neocortices. Examination of murine and human neocortex reveals that the RNA binding protein and translational regulator, CELF4, is expressed in compartments enriched in initial synaptogenesis: the marginal zone and the subplate. We also find that Celf4/CELF4-target mRNAs are encoded by risk genes for adverse neurodevelopmental outcomes translating into synaptic proteins. Surprisingly, deleting Celf4 in the forebrain disrupts the balance of subplate synapses in a sex-specific fashion. This highlights the significance of RNA binding proteins and mRNA translation in evolutionarily advanced synaptic development, potentially contributing to sex differences.

Role of CELF2 in ferroptosis: Potential targets for cancer therapy (Review).

Ferroptosis is a novel form of regulated cellular necrosis that plays a critical role in promoting cancer progression and developing drug resistance. The main characteristic of ferroptosis is iron­dependent lipid peroxidation caused by excess intracellular levels of reactive oxygen species. CUGBP ELAV­like family number 2 (CELF2) is an RNA­binding protein that is downregulated in various types of cancer and is associated with poor patient prognoses. CELF2 can directly bind mRNA to a variety of ferroptosis control factors; however, direct evidence of the regulatory role of CELF2 in ferroptosis is currently limited. The aim of the present review was to summarise the findings of previous studies on CELF2 and its role in regulating cellular redox homeostasis. The present review may provide insight into the possible mechanisms through which CELF2 affects ferroptosis and to provide recommendations for future studies.

Long noncoding RNA LINC01594 inhibits the CELF6-mediated splicing of oncogenic CD44 variants to promote colorectal cancer metastasis.

Long noncoding RNAs (lncRNAs) play critical roles in tumorigenesis and tumor metastasis. However, the underlying mechanisms of lncRNAs in colorectal cancer (CRC) need further exploration. By using data from The Cancer Genome Atlas (TCGA) and GEO databases, we identified a novel CRC-related lncRNA, LINC01594, that is significantly upregulated in CRC and associated with poor prognosis. In vitro and in vivo, gain- and loss-of-function experiments demonstrated that LINC01594 promotes metastasis in CRC. LINC01594 functions as a DNMT1 scaffold, increasing the level of CELF6 promoter methylation. LINC01594 also competitively binds the transcription factor p53, decreasing CELF6 expression. This inhibited the exon skipping of CD44 V4-V7 induced by CELF6. In summary, this study highlights a novel CRC biomarker and therapeutic target, LINC01594, and the findings suggest that the LINC01594-CELF6-CD44 axis might serve as a biomarker and therapeutic target in CRC.

Hypoxia-induced lncRNA MRVI1-AS1 accelerates hepatocellular carcinoma progression by recruiting RNA-binding protein CELF2 to stabilize SKA1 mRNA.

BACKGROUND: Long non-coding RNAs (lncRNAs) perform a vital role during the progression of hepatocellular carcinoma (HCC). Here, we aimed to identify a novel lncRNA involved in HCC development and elucidate the underlying molecular mechanism. METHODS: The RT-qPCR and TCGA dataset analysis were applied to explore the expressions of MRVI1-AS1 in HCC tissues and cell lines. Statistical analysis was applied to analyze the clinical significance of MRVI1-AS1 in HCC. The functions of MRVI1-AS1 in HCC cells metastasis and growth were explored by transwell assays, wound healing assay, MTT assay, EdU assay, the intravenous transplantation tumor model, and the subcutaneous xenograft tumor model. Microarray mRNA expression analysis, dual luciferase assays, and actinomycin D treatment were used to explore the downstream target of MRVI1-AS1 in HCC cells. RIP assay was applied to assess the direct interactions between CELF2 and MRVI1-AS1 or SKA1 mRNA. Rescue experiments were employed to validate the functional effects of MRVI1-AS1, CELF2, and SKA1 on HCC cells. RESULTS: MRVI1-AS1 was found to be dramatically upregulated in HCC and the expression was strongly linked to tumor size, venous infiltration, TNM stage, as well as HCC patients' outcome. Cytological experiments and animal experiments showed that MRVI1-AS1 promoted HCC cells metastasis and growth. Furthermore, SKA1 was identified as the downstream targeted mRNA of MRVI1-AS1 in HCC cells, and MRVI1-AS1 increased SKA1 expression by recruiting CELF2 protein to stabilize SKA1 mRNA. In addition, we found that MRVI1-AS1 expression was stimulated by hypoxia through a HIF-1-dependent manner, which meant that MRVI1-AS was a direct downstream target gene of HIF-1 in HCC. CONCLUSION: In a word, our findings elucidated that hypoxia-induced MRVI1-AS1 promotes metastasis and growth of HCC cells via recruiting CELF2 protein to stabilize SKA1 mRNA, pointing to MRVI1-AS1 as a promising clinical application target for HCC therapy.

MIMT1 and LINC01550 are uncharted lncRNAs down-regulated in colorectal cancer.

Incomplete knowledge of the molecular basis of colorectal cancer, with subsequent limitations in early diagnosis and effective treatment, has contributed to this form of malignancy becoming the second most common cause of cancer-related death worldwide. With the advances in high-throughput profiling techniques and the availability of public data sets such as The Cancer Genome Atlas Program (TCGA), a broad range of coding transcripts have been profiled and their underlying modes of action have been mapped. However, there is still a huge gap in our understanding of noncoding RNA dysregulation. To this end, we used a bioinformatics approach to shortlist and evaluate yet-to be-profiled long noncoding RNAs (lncRNAs) in colorectal cancer. We analysed the TCGA RNA-seq data and followed this by validating the expression patterns using a qPCR technique. Analysing in-house clinical samples, the real-time PCR method revealed that the shortlisted lncRNAs, that is MER1 Repeat Containing Imprinted Transcript 1 (MIMT1) and Non-Protein Coding RNA 1550 (LINC01550), were down-regulated in colorectal cancer tumours compared with the paired adjacent normal tissues. Mechanistically, the in silico results suggest that LINC01550 could form a complex competitive endogenous RNA (ceRNA) network leading to the subsequent regulation of colorectal cancer-related genes, such as CUGBP Elav-Like Family Member (CELF2), Polypyrimidine Tract Binding Protein 1 (PTBP1) and ELAV Like RNA Binding Protein 1 (ELAV1). The findings of this work indicate that MIMT1 and LINC01550 could be novel tumour suppressor genes that can be studied further to assess their roles in regulating the cancer signalling pathway(s).

Role of SNPs located in the exon 9 of ATAPA1 gene on goose egg production.

The meat and egg of goose is one of the main components of human food supply. The improvement of goose egg production is particularly important for the increasing human population. However, limited information is available about the effective molecular markers and mechanisms of egg production in goose. In this study, we jointly utilized the data of genome resequencing in different egg production Sichuan white goose and transcriptome at different follicle development stages to identified the molecular markers and mechanisms of egg production. The coefficient of variation of individual egg production in Sichuan white goose population is 0.42 to 0.49. Fifty individuals with the highest (laying 365 days egg number, LEN365 = 79-145) and 50 individuals with the lowest (LEN365 = 8-48) egg production were divided into high and low egg production groups. Based on whole-genome sequencing data of the selected samples, 36 SNPs (annotation novel.12.470, CELF2, ATP1A1, KCNJ6, RAB4A, UST, REV3L, DHX15, CAVN2, SLC5A9, Cldn5, MRPS23, and Tspan2) associated with the LEN365 were identified, involving multiple pathways such as metabolism and endocrinology. Notably, 5 SNPs located in the exon9 of ATP1A1 were identified by GWAS analysis. The association analysis with LEN365 showed the phenotypic variance explained of this haplotype consisting of 5 SNPs is 20.51%. Through transcriptome data analysis, we found the expression of ATP1A1 in the granular layers was increased in the stage of small yellow follicle to large yellow follicle (LYF) and LYF to F5, while decreased in F2 to F1. For the first time, we report the haplotype region formed by 5 SNPS on exon9 of ATP1A1 is associated with egg production in goose and involved in follicle selection and maturation processes.

A Combination of Bio-Orthogonal Supramolecular Clicking and Proximity Chemical Tagging as a Supramolecular Tool for Discovery of Putative Proteins Associated with Laminopathic Disease.

Protein mutations alter protein-protein interactions that can lead to a number of illnesses. Mutations in lamin A (LMNA) have been reported to cause laminopathies. However, the proteins associated with the LMNA mutation have mostly remained unexplored. Herein, a new chemical tool for proximal proteomics is reported, developed by a combination of proximity chemical tagging and a bio-orthogonal supramolecular latching based on cucurbit[7]uril (CB[7])-based host-guest interactions. As this host-guest interaction acts as a noncovalent clickable motif that can be unclicked on-demand, this new chemical tool is exploited for reliable detection of the proximal proteins of LMNA and its mutant that causes laminopathic dilated cardiomyopathy (DCM). Most importantly, a comparison study reveals, for the first time, mutant-dependent alteration in LMNA proteomic environments, which allows to identify putative laminopathic DCM-linked proteins including FOXJ3 and CELF2. This study demonstrates the feasibility of this chemical tool for reliable proximal proteomics, and its immense potential as a new research platform for discovering biomarkers associated with protein mutation-linked diseases.

M2 macrophage-derived exosomal miR-1911-5p promotes cell migration and invasion in lung adenocarcinoma by down-regulating CELF2 -activated ZBTB4 expression.

Lung adenocarcinoma (LUAD) is one of the most aggressive, lethal cancers, comprising around 40% of lung cancer cases. Metastases are the primary cause of LUAD deaths. The mechanism underlying metastatic LUAD and tumor microenvironment remain largely unknown. To explore the effect of M2 macrophage-derived exosomes on LUAD progression. Quantitative-PCR (q-PCR) and western blot were used to measure the expression of RNAs and proteins separately. Co-culture experiments wound healing and Transwell invasion assays were performed to evaluate the effect of M2 macrophage-derived exosomes on LUAD cell migration and invasion. RNA pulldown and luciferase reporter, RNA-binding immunoprecipitation (RIP), and mRNA stability assays were conducted to explore the downstream mechanism of exosomal microRNA-1911-5p (miR-1911-5p). M2 macrophage-derived exosomes accelerated the migration and invasion of LUAD cells. M2 macrophages-secreted exosomal miR-1911-5p enhanced cell migration and invasion in LUAD. Mechanically, miR-1911-5p targeted CUGBP- and ETR-3-like family 2 (CELF2) to downregulate zinc finger and BTB domain containing 4 (ZBTB4) in LUAD. Additionally, miR-1911-5p promoted LUAD progression via ZBTB4. The present study demonstrated that M2 macrophage-derived exosomal miR-1911-5p facilitates the migration and invasion of LUAD cells by inhibiting CELF2-activated ZBTB4, which might offer insight into LUAD treatment.

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.

Exosomal miR-625-3p secreted by cancer-associated fibroblasts in colorectal cancer promotes EMT and chemotherapeutic resistance by blocking the CELF2/WWOX pathway.

Migration, invasion, epithelial-mesenchymal transformation (EMT), and chemotherapeutic resistance are the leading causes of therapeutic failure in people with colorectal cancer (CRC). The migration of exosomal miRNA between cancer cells and the tumor microenvironment is directly associated with malignant behavior in cancer-associated fibroblasts (CAFs). In the context of earlier research, the purpose of the current study was to assess the role and potential mechanism of miR-625-3p released by CAFs in CRC cells. Exosomes were extracted and purified from CAFs conditioned medium by ultracentrifugation. Western blot, immunohistochemistry, CCK-8, transwell assay, H&E staining, Tunnel, real-time PCR, double luciferase assay, RNA-binding protein immunoprecipitation (RIP), and immunofluorescence double staining experiments were used to investigate the effects of CAFs-Exo and miR-625-3p on CRC cell invasion, migration, proliferation, EMT, chemotherapeutic resistance, and molecular mechanisms. The current results indicated that CAFs-Exo was directly internalized by CRC cells, and exosomal miR-625-3p derived from CAFs might promote migration, invasion, EMT and chemotherapeutic resistance in CRC cells by inhibiting the CELF2/WWOX pathway, providing a potential candidate for CRC prediction and treatment.

Natural Antisense Long Noncoding RNA HHIP-AS1 Suppresses Non-Small-Cell Lung Cancer Progression by Increasing HHIP Stability via Interaction with CELF2.

Non-small-cell lung cancer (NSCLC) is a major category of lung cancer, with high incidence and high mortality. Natural antisense long noncoding RNAs (lncRNAs) are involved in the development of NSCLC via their regulation of biological processes. However, the function of the lncRNA Hedgehog-interacting protein antisense RNA 1 (HHIP-AS1) in NSCLC is mostly unknown. In the study discussed here, HHIP-AS1 and HHIP levels were predicted based on the TCGA database, and detected via qRT-PCR or western blotting assays. Cell proliferation, migration, and invasion were measured via CCK-8 and trans-well assays. Related protein levels were measured using western blotting analysis. The results showed that HHIP-AS1 and HHIP levels are downregulated in NSCLC, and that low HHIP-AS1 and HHIP expression is associated with poor outcomes. HHIP-AS1 overexpression represses cell proliferation, migration, and invasion in NSCLC. HHIP-AS1 enhances HHIP expression and stability, and this effect is mediated by CELF2. HHIP silencing attenuates the suppressive roles of HHIP-AS1 in proliferation, migration, and invasion. As a result of these findings, it is concluded that HHIP-AS1 overexpression restrains proliferation, migration, and invasion of NSCLC cells by increasing HHIP stability via its targeting of CELF2.

Construction of MicroRNA-Target Interaction Networks Based on MicroRNA Expression Profiles of HRV16-infected H1-HeLa Cells.

In the present study we investigated the changes in miRNA levels inhuman rhinovirus 16 (HRV16)-infected cells. A small RNA deep sequencing experiment was performed through next-generation sequencing. In total, 53 differentially expressed miRNAs were confirmed by RT-qPCR, including 37 known miRNAs and 16 novel miRNAs. Interaction networks between differentially expressed miRNAs and their targets were established by mirDIP and Navigator. The prediction results showed that QKI, NFAT5, BNC2, CELF2, LCOR, MBNL2, MTMR3, NFIB, PPARGC1A, RSBN1, TRPS1, WDR26, and ZNF148, which are associated with cellular differentiation and transcriptional regulation, were recognized by 12, 11, or 9 miRNAs. Many correlations were observed between transcriptional or post-transcriptional regulation of an miRNA and the expression levels of its target genes in HRV16-infected H1-HeLa cells.

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.

RNA-binding protein CELF6 modulates transcription and splicing levels of genes associated with tumorigenesis in lung cancer A549 cells.

CELF6 (CUGBP Elav-Like Family Member 6), a canonical RNA binding protein (RBP), plays important roles in post-transcriptional regulation of pre-mRNAs. However, the underlying mechanism of lower expressed CELF6 in lung cancer tissues is still unclear. In this study, we increased CELF6 manually in lung cancer cell line (A549) and utilized transcriptome sequencing (RNA-seq) technology to screen out differentially expressed genes (DEGs) and alternative splicing events (ASEs) after CELF6 over-expression (CELF6-OE). We found that CELF6-OE induced 417 up-regulated and 1,351 down-regulated DEGs. Functional analysis of down-regulated DEGs showed that they were highly enriched in immune/inflammation response- related pathways and cell adhesion molecules (CAMs). We also found that CELF6 inhibited the expression of many immune-related genes, including TNFSF10, CCL5, JUNB, BIRC3, MLKL, PIK3R2, CCL20, STAT1, MYD88, and CFS1, which mainly promote tumorigenesis in lung cancer. The dysregulated DEGs were also validated by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) experiment. In addition, CELF6 regulates the splicing pattern of large number of genes that are enriched in p53 signaling pathway and apoptosis, including TP53 and CD44. In summary, we made an extensive analysis of the transcriptome profile of gene expression and alternative splicing by CELF6-OE, providing a global understanding of the target genes and underlying regulation mechanisms mediated by CELF6 in the pathogenesis and development of lung cancer.

MECP2-related pathways are dysregulated in a cortical organoid model of myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is a multisystem, autosomal-dominant inherited disorder caused by CTG microsatellite repeat expansions (MREs) in the 3' untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene. Despite its prominence as the most common adult-onset muscular dystrophy, patients with congenital to juvenile-onset forms of DM1 can present with debilitating neurocognitive symptoms along the autism spectrum, characteristic of possible in utero cortical defects. However, the molecular mechanism by which CTG MREs lead to these developmental central nervous system (CNS) manifestations is unknown. Here, we showed that CUG foci found early in the maturation of three-dimensional (3D) cortical organoids from DM1 patient-derived induced pluripotent stem cells (iPSCs) cause hyperphosphorylation of CUGBP Elav-like family member 2 (CELF2) protein. Integrative single-cell RNA sequencing and enhanced cross-linking and immunoprecipitation (eCLIP) analysis revealed that reduced CELF2 protein-RNA substrate interactions results in misregulation of genes critical for excitatory synaptic signaling in glutamatergic neurons, including key components of the methyl-CpG binding protein 2 (MECP2) pathway. Comparisons to MECP2(y/-) cortical organoids revealed convergent molecular and cellular defects such as glutamate toxicity and neuronal loss. Our findings provide evidence suggesting that early-onset DM1 might involve neurodevelopmental disorder-associated pathways and identify N-methyl-d-aspartic acid (NMDA) antagonists as potential treatment avenues for neuronal defects in DM1.

Screening for Immune-Related RNA Biomarkers of Aneurysmal Subarachnoid Hemorrhage.

PURPOSE: Through comprehensive bioinformatics analysis based on the immune microenvironment, this study aimed to identify immune-related RNA biomarkers that indicate aneurysmal subarachnoid hemorrhage (aSAH). METHODS: The GSE73378 dataset was downloaded from the National Center for Biotechnology Information GEO database, providing blood from 107 normal controls and 103 patients with aSAH. The immune infiltration types in the aSAH blood samples were assessed and RNAs that were differentially expressed (DE) between 1) the aSAH and control groups and 2) the immune infiltration groups (high and low) were identified. The intersecting genes were subjected to weighted gene co-expression network analysis followed by co-expression network construction. The aSAH-related genes and pathways were identified from the Comparative Toxicogenomics Database: update 2019. RESULTS: A total of three DE long non-coding RNAs (lncRNAs) and 301 DE mRNAs were identified. Of the 301 mRNAs, 91 were significantly enriched in three modules. Based on the 91 mRNAs and three lncRNAs, a co-expression network related to the disease pathway was constructed. This pathway consisted of 16 factors, including the 13 mRNAs (e.g., TNFSF13B, TNFSF10, MYD88, GNA12 and NSMAF) and three lncRNAs (FAM66A, LINC00954 and CELF2-AS2), as well as six pathways, including the NF-κB, toll-like receptor, and sphingolipid signalling pathways. CONCLUSION: TNFSF13B, MYD88, GNA12, NSMAF, FAM66A, LINC00954 and CELF2-AS2 may serve as biomarkers for aSAH. The NF-κB, toll-like receptor and sphingolipid signalling pathways may play critical roles in the progression of aSAH.

Methylated DNA markers for plasma detection of ovarian cancer: Discovery, validation, and clinical feasibility.

OBJECTIVE: Aberrant DNA methylation is an early event in carcinogenesis which could be leveraged to detect ovarian cancer (OC) in plasma. METHODS: DNA from frozen OC tissues, benign fallopian tube epithelium (FTE), and buffy coats from cancer-free women underwent reduced representation bisulfite sequencing (RRBS) to identify OC MDMs. Candidate MDM selection was based on receiver operating characteristic (ROC) discrimination, methylation fold change, and low background methylation among controls. Blinded biological validation was performed using methylated specific PCR on DNA extracted from independent OC and FTE FFPE tissues. MDMs were tested using Target Enrichment Long-probe Quantitative Amplified Signal (TELQAS) assays in pre-treatment plasma from women newly diagnosed with OC and population-sampled healthy women. A random forest modeling analysis was performed to generate predictive probability of disease; results were 500-fold in silico cross-validated. RESULTS: Thirty-three MDMs showed marked methylation fold changes (10 to >1000) across all OC subtypes vs FTE. Eleven MDMs (GPRIN1, CDO1, SRC, SIM2, AGRN, FAIM2, CELF2, RIPPLY3, GYPC, CAPN2, BCAT1) were tested on plasma from 91 women with OC (73 (80%) high-grade serous (HGS)) and 91 without OC; the cross-validated 11-MDM panel highly discriminated OC from controls (96% (95% CI, 89-99%) specificity; 79% (69-87%) sensitivity, and AUC 0.91 (0.86-0.96)). Among the 5 stage I/II HGS OCs included, all were correctly identified. CONCLUSIONS: Whole methylome sequencing, stringent filtering criteria, and biological validation yielded candidate MDMs for OC that performed with high sensitivity and specificity in plasma. Larger plasma-based OC MDM studies, including testing of pre-diagnostic specimens, are warranted.