MeCP2 gene therapy ameliorates disease phenotype in mouse model for Pitt Hopkins syndrome.

The neurodevelopmental disorder Pitt Hopkins syndrome (PTHS) causes clinical symptoms similar to Rett syndrome (RTT) patients. However, RTT is caused by MECP2 mutations whereas mutations in the TCF4 gene lead to PTHS. The mechanistic commonalities underling these two disorders are unknown, but their shared symptomology suggest that convergent pathway-level disruption likely exists. We reprogrammed patient skin derived fibroblasts into induced neuronal progenitor cells. Interestingly, we discovered that MeCP2 levels were decreased in PTHS patient iNPCs relative to healthy controls and that both iNPCs and iAstrocytes displayed defects in function and differentiation in a mutation-specific manner. When Tcf4+/- mice were genetically crossed with mice overexpressing MeCP2, molecular and phenotypic defects were significantly ameliorated, underlining and important role of MeCP2 in PTHS pathology. Importantly, post-natal intracerebroventricular gene replacement therapy with adeno-associated viral vector serotype 9 (AAV9)-expressing MeCP2 (AAV9.P546.MeCP2) significantly improved iNPC and iAstrocyte function and effectively ameliorated histological and behavioral defects in Tcf4+/- mice. Combined, our data suggest a previously unknown role of MeCP2 in PTHS pathology and common pathways that might be affected in multiple neurodevelopmental disorders. Our work highlights potential novel therapeutic targets for PTHS, including upregulation of MeCP2 expression or its downstream targets or, potentially, MeCP2-based gene therapy.

Expanding the phenotype in Pitt-Hopkins syndrome; description of new oral finding and dental management considerations.

BACKGROUND: Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder with physical, cognitive, and behavioral characteristics that is caused by heterozygous mutations in the TCF4 gene. Patients with PTHS might present a unique challenge for oral healthcare professionals because of the associated comorbidities. CASE REPORT: Here we describe a new case of PTHS in a 13-year-old girl with particular emphasis on oro-dental findings and oral healthcare management. Observed oro-dental findings in our case included shallow palate, absence of lingual frenum, gingival enlargement, thick lips and relative microdontia. The patient was unable to tolerate dental care under local anesthesia. Therefore, comprehensive dental treatment was performed under general anesthesia after a careful pre-anesthetic cardio-respiratory, neurological, and hematological evaluation. The patient was closely monitored intra-operatively for breathing rhythm, O2 saturation, and signs of respiratory distress. The patient was observed for 24 h post-op for respiratory distress and was discharged then uneventfully. CONCLUSION: Dental treatment under general anesthesia in these patients might be complicated by the abnormal breathing rhythm, and close monitoring and follow up for signs of respiratory distress after general anesthesia is necessary. Recognition of oral and dental findings might help to expand the phenotype and better characterize rare syndromes.

Dickkopf-1 (DKK1) blockade mitigates osteogenesis imperfecta (OI) related bone disease.

BACKGROUND: The current treatment of osteogenesis imperfecta (OI) is imperfect. Our study thus delves into the potential of using Dickkopf-1 antisense (DKK1-AS) to treat OI. METHODS: We analysed serum DKK1 levels and their correlation with lumbar spine and hip T-scores in OI patients. Comparative analyses were conducted involving bone marrow stromal cells (BMSCs) and bone tissues from wild-type mice, untreated OI mice, and OI mice treated with DKK1-ASor DKK1-sense (DKK1-S). RESULTS: Significant inverse correlations were noted between serum DKK1 levels and lumbar spine (correlation coefficient = - 0.679, p = 0.043) as well as hip T-scores (correlation coefficient = - 0.689, p = 0.042) in OI patients. DKK1-AS improved bone mineral density (p = 0.002), trabecular bone volume/total volume fraction (p < 0.001), trabecular separation (p = 0.010), trabecular thickness (p = 0.001), trabecular number (p < 0.001), and cortical thickness (p < 0.001) in OI mice. DKK1-AS enhanced the transcription of collagen 1α1, osteocalcin, runx2, and osterix in BMSC from OI mice (all p < 0.001), resulting in a higher von Kossa-stained matrix area (p < 0.001) in ex vivo osteogenesis assays. DKK1-AS also reduced osteoclast numbers (p < 0.001), increased ß-catenin and T-cell factor 4 immunostaining reactivity (both p < 0.001), enhanced mineral apposition rate and bone formation rate per bone surface (both p < 0.001), and decreased osteoclast area (p < 0.001) in OI mice. DKK1-AS upregulated osteoprotegerin and downregulated nuclear factor-kappa B ligand transcription (both p < 0.001). Bone tissues from OI mice treated with DKK1-AS exhibited significantly higher breaking force compared to untreated OI mice (p < 0.001). CONCLUSIONS: Our study elucidates that DKK1-AS has the capability to enhance bone mechanical properties, restore the transcription of osteogenic genes, promote osteogenesis, and inhibit osteoclastogenesis in OI mice.

DNA methylation episignature and comparative epigenomic profiling for Pitt-Hopkins syndrome caused by TCF4 variants.

Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder caused by pathogenic variants in TCF4, leading to intellectual disability, specific morphological features, and autonomic nervous system dysfunction. Epigenetic dysregulation has been implicated in PTHS, prompting the investigation of a DNA methylation (DNAm) "episignature" specific to PTHS for diagnostic purposes and variant reclassification and functional insights into the molecular pathophysiology of this disorder. A cohort of 67 individuals with genetically confirmed PTHS and three individuals with intellectual disability and a variant of uncertain significance (VUS) in TCF4 were studied. The DNAm episignature was developed with an Infinium Methylation EPIC BeadChip array analysis using peripheral blood cells. Support vector machine (SVM) modeling and clustering methods were employed to generate a DNAm classifier for PTHS. Validation was extended to an additional cohort of 11 individuals with PTHS. The episignature was assessed in relation to other neurodevelopmental disorders and its specificity was examined. A specific DNAm episignature for PTHS was established. The classifier exhibited high sensitivity for TCF4 haploinsufficiency and missense variants in the basic-helix-loop-helix domain. Notably, seven individuals with TCF4 variants exhibited negative episignatures, suggesting complexities related to mosaicism, genetic factors, and environmental influences. The episignature displayed degrees of overlap with other related disorders and biological pathways. This study defines a DNAm episignature for TCF4-related PTHS, enabling improved diagnostic accuracy and VUS reclassification. The finding that some cases scored negatively underscores the potential for multiple or nested episignatures and emphasizes the need for continued investigation to enhance specificity and coverage across PTHS-related variants.

Tcf4 dysfunction alters dorsal and ventral cortical neurogenesis in Pitt-Hopkins syndrome mouse model showing sexual dimorphism.

Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder caused by haploinsufficiency of transcription factor 4 (TCF4). In this work, we focused on the cerebral cortex and investigated in detail the progenitor cell dynamics and the outcome of neurogenesis in a PTHS mouse model. Labeling and quantification of progenitors and newly generated neurons at various time points during embryonic development revealed alterations affecting the dynamic of cortical progenitors since the earliest stages of cortex formation in PTHS mice. Consequently, establishment of neuronal populations and layering of the cortex were found to be altered in heterozygotes subjects at birth. Interestingly, defective layering process of pyramidal neurons was partially rescued by reintroducing TCF4 expression using focal in utero electroporation in the cerebral cortex. Coincidentally with a defective dorsal neurogenesis, we found that ventral generation of interneurons was also defective in this model, which may lead to an excitation/inhibition imbalance in PTHS. Overall, sex-dependent differences were detected with more marked effects evidenced in males compared with females. All of this contributes to expand our understanding of PTHS, paralleling the advances of research in autism spectrum disorder and further validating the PTHS mouse model as an important tool to advance preclinical studies.

AMD1 promotes breast cancer aggressiveness via a spermidine-eIF5A hypusination-TCF4 axis.

BACKGROUND: Basal-like breast cancer (BLBC) is the most aggressive subtype of breast cancer due to its aggressive characteristics and lack of effective therapeutics. However, the mechanism underlying its aggressiveness remains largely unclear. S-adenosylmethionine decarboxylase proenzyme (AMD1) overexpression occurs specifically in BLBC. Here, we explored the potential molecular mechanisms and functions of AMD1 promoting the aggressiveness of BLBC. METHODS: The potential effects of AMD1 on breast cancer cells were tested by western blotting, colony formation, cell proliferation assay, migration and invasion assay. The spermidine level was determined by high performance liquid chromatography. The methylation status of CpG sites within the AMD1 promoter was evaluated by bisulfite sequencing PCR. We elucidated the relationship between AMD1 and Sox10 by ChIP assays and quantitative real-time PCR. The effect of AMD1 expression on breast cancer cells was evaluated by in vitro and in vivo tumorigenesis model. RESULTS: In this study, we showed that AMD1 expression was remarkably elevated in BLBC. AMD1 copy number amplification, hypomethylation of AMD1 promoter and transcription activity of Sox10 contributed to the overexpression of AMD1 in BLBC. AMD1 overexpression enhanced spermidine production, which enhanced eIF5A hypusination, activating translation of TCF4 with multiple conserved Pro-Pro motifs. Our studies showed that AMD1-mediated metabolic system of polyamine in BLBC cells promoted tumor cell proliferation and tumor growth. Clinically, elevated expression of AMD1 was correlated with high grade, metastasis and poor survival, indicating poor prognosis of breast cancer patients. CONCLUSION: Our work reveals the critical association of AMD1-mediated spermidine-eIF5A hypusination-TCF4 axis with BLBC aggressiveness, indicating potential prognostic indicators and therapeutic targets for BLBC.

miR-140-y targets TCF4 to regulate the Wnt signaling pathway and promote embryonic feather follicle development in Hungarian white goose.

Goose down feather has become one of the most important economical products in the goose breeding industry and it provides several essential physiological roles in birds. Therefore, understanding and regulating the development of skin and feather follicles during embryogenesis is critical for avian biology and the poultry industry. MicroRNAs are known to play an important role in controlling gene expression during skin and feather follicle development. In this study, bioinformatics analysis was conducted to select miR-140-y as a potential miRNA involved in skin and feather follicle development and to predict TCF4 as its target gene. This gene was expressed at significant levels during embryonic feather follicle development, as identified by qPCR and Western blot. The targeting relationship was confirmed by a dual-luciferase assay in 293T cells. Then, the miR-140-y/TCF4 function in dermal fibroblast cells was explored. The results showed that miR-140-y could suppress the proliferation of goose embryonic dermal fibroblast cells (GEDFs) by suppressing the activity of some Wingless-types (Wnt) pathway related genes and proliferation marker genes, while miR-140-y inhibition led to the opposite effect. Similarly, the inhibition of the TCF4 gene results in blocking the proliferation of GEDFs by reducing the activity of some Wnt pathway-related genes. Finally, the co-transfection of miR-140-y inhibitor and siRNA-TCF4 results in a rescue of the TCF4 function and an increase of the Wnt signaling pathway and GEDFs proliferation. In conclusion, these results demonstrated that the miR-140-y-TCF4 axis influences the activity of the Wnt signaling pathway and works as a dynamic regulator during skin and feather follicle development.

ß-Catenin regulates ovarian granulosa cell cycle and proliferation in laying hens by interacting with TCF4.

Ovarian follicle development depends on the proliferation and differentiation of granulosa cells and is a complex biological process. The Wnt/ß-catenin signaling pathway can regulate ovarian follicle development, and ß-catenin, encoded by catenin beta 1 (CTNNB1), is the core component of this pathway. Although several studies of the mechanisms by which the Wnt/ß-catenin pathway regulates cell proliferation in humans and mammals have reported, it remains unclear how ß-catenin functions in poultry. To investigate the function of ß-catenin in laying hens' follicle development, we evaluated the effect of CTNNB1 on cell cycle, proliferation, and apoptosis in ovarian granulosa cells (GCs) isolated from laying hens. We demonstrated that CTNNB1 significantly affected the expression of cyclin D1 (CCND1) and v-myc avian myelocytomatosis viral oncogene homolog (c-Myc) (P < 0.01 and P < 0.05), key genes related to cell cycle and proliferation, to promote cell cycle progression from G1 to S phase, and thus accelerate granulosa cell proliferation. CTNNB1 did not however affect apoptosis or the expression of related genes baculoviral IAP repeat containing 5 (BIRC5) and BCL2 apoptosis regulator (Bcl-2). Overexpression of transcription factor 7-like 2 (TCF4) resulted in increased expression of CCND1, accelerated cell cycle progression, and granulosa cell proliferation. Direct physical interaction between ß-catenin and TCF4 was demonstrated by immunofluorescence and coimmunoprecipitation. The proliferation of granulosa cells was inhibited by silencing CCND1; overexpression of TCF4 in CCND1-silenced cells restored their proliferation rate to normal levels. These results indicate that the interaction of TCF4 and ß-catenin promotes CCND1 expression which in turn accelerates the cell cycle process of laying hen hierarchical follicular granulosa cells.

Chromatin and DNA Dynamics in Mouse Models of Liver Cancers.

In recent years, important efforts have been made to understand how the expression of a specific gene repertoire correlates with chromatin accessibility, histone mark deposition, as well as with chromatin looping establishing connectivity with regulatory regions. The emergence of new techniques for genome-wide analyses and their progressive optimization to work on low amounts of material allows the scientific community to obtain an integrated view of transcriptional landscapes in physiology and disease. Here, we describe our own experience aiming at correlating the TCF-4/ß-catenin cistrome during liver tumorigenesis with chromatin remodeling, histone mark modifications, and long-distance DNA looping.

Clinical and genetic characterization of 47 Chinese pediatric patients with Pitt-Hopkins syndrome: a retrospective study.

BACKGROUND: Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder that remains underdiagnosed and its clinical presentations and mutation profiles in a diverse population are yet to be evaluated. This retrospective study aims to investigate the clinical and genetic characteristics of Chinese patients with PTHS. METHODS: The clinical, biochemical, genetic, therapeutic, and follow-up data of 47 pediatric patients diagnosed with PTHS between 2018 and 2021 were retrospectively analyzed. RESULTS: The Chinese PTHS patients presented with specific facial features and exhibited global developmental delay of wide severity range. The locus heterogeneity of the TCF4 gene in the patients was highlighted, emphasizing the significance of genetic studies for accurate diagnosis, albeit no significant correlations between genotype and phenotype were observed in this cohort. The study also reports the outcomes of patients who underwent therapeutic interventions, such as ketogenic diets and biomedical interventions. CONCLUSIONS: The findings of this retrospective analysis expand the phenotypic and molecular spectra of PTHS patients. The study underscores the need for a long-term prospective follow-up study to assess potential therapeutic interventions.

CTG18.1 expansion in transcription factor 4 (TCF4) in corneal graft failure: preliminary study.

Fuchs endothelial corneal dystrophy (FECD) is caused by a corneal endothelial cell loss, leading to corneal edema and visual impairment. The most significant genetic risk factor for FECD is an expansion of the CTG18.1 locus in transcription factor 4 (TCF4). The current treatment for severe FECD is corneal transplantation, with Descemet stripping automated keratoplasty (DSAEK) as a common surgical method. Although successful in most cases, the risk for transplant failure due to diverse causes must be considered. In this study, we investigated if presence of TCF4 CTG18.1 expansion with more than 31 (n ≥ 31) repeats in donated corneal grafts could be a reason for corneal transplant failure after DSAEK. For this, nine consecutively failed DSAEK corneal grafts were genotyped for CTG18.1 repeat length. One-sided Mann-Whitney U test was performed to evaluate if failed DSAEK corneal grafts had longer CTG18.1 repeats than healthy controls from the same population. All failed corneal grafts had CTG18.1 n ≤ 27 with a median of 18 (IQR 8.0) repeats for the longest allele. There was no statistical difference in CTG18.1 repeat lengths between failed corneal grafts and the geographically matched healthy control group. In conclusion, none of the nine failed corneal grafts in our material had CTG18.1 repeat lengths ≥ 31, a cut-off known to have a biological relevance in FECD. Thus, our results suggest that the assessment of donors and inspection of the corneal tissue before the decision for procurement is sufficient, in terms of recognizing FECD in the donor.

A TCF4-dependent gene regulatory network confers resistance to immunotherapy in melanoma.

To better understand intrinsic resistance to immune checkpoint blockade (ICB), we established a comprehensive view of the cellular architecture of the treatment-naive melanoma ecosystem and studied its evolution under ICB. Using single-cell, spatial multi-omics, we showed that the tumor microenvironment promotes the emergence of a complex melanoma transcriptomic landscape. Melanoma cells harboring a mesenchymal-like (MES) state, a population known to confer resistance to targeted therapy, were significantly enriched in early on-treatment biopsies from non-responders to ICB. TCF4 serves as the hub of this landscape by being a master regulator of the MES signature and a suppressor of the melanocytic and antigen presentation transcriptional programs. Targeting TCF4 genetically or pharmacologically, using a bromodomain inhibitor, increased immunogenicity and sensitivity of MES cells to ICB and targeted therapy. We thereby uncovered a TCF4-dependent regulatory network that orchestrates multiple transcriptional programs and contributes to resistance to both targeted therapy and ICB in melanoma.

TCF4 Mutations Disrupt Synaptic Function Through Dysregulation of RIMBP2 in Patient-Derived Cortical Neurons.

BACKGROUND: Genetic variation in the TCF4 (transcription factor 4) gene is associated with risk for a variety of developmental and psychiatric conditions, which includes a syndromic form of autism spectrum disorder called Pitt-Hopkins syndrome (PTHS). TCF4 encodes an activity-dependent transcription factor that is highly expressed during cortical development and in animal models has been shown to regulate various aspects of neuronal development and function. However, our understanding of how disease-causing mutations in TCF4 confer pathophysiology in a human context is lacking. METHODS: To model PTHS, we differentiated human cortical neurons from human induced pluripotent stem cells that were derived from patients with PTHS and neurotypical individuals. To identify pathophysiology and disease mechanisms, we assayed cortical neurons with whole-cell electrophysiology, Ca2+ imaging, multielectrode arrays, immunocytochemistry, and RNA sequencing. RESULTS: Cortical neurons derived from patients with TCF4 mutations showed deficits in spontaneous synaptic transmission, network excitability, and homeostatic plasticity. Transcriptomic analysis indicated that these phenotypes resulted in part from altered expression of genes involved in presynaptic neurotransmission and identified the presynaptic binding protein RIMBP2 as the most differentially expressed gene in PTHS neurons. Remarkably, TCF4-dependent deficits in spontaneous synaptic transmission and network excitability were rescued by increasing RIMBP2 expression in presynaptic neurons. CONCLUSIONS: Taken together, these results identify TCF4 as a critical transcriptional regulator of human synaptic development and plasticity and specifically identifies dysregulation of presynaptic function as an early pathophysiology in PTHS.

Nanopore sequencing method for CTG18.1 expansion in TCF4 in late-onset Fuchs endothelial corneal dystrophy and a comparison of the structural features of cornea with first-degree relatives.

BACKGROUND: To evaluate the relationship between the number of trinucleotide repeats (TNR) in late-onset Fuchs corneal endothelial dystrophy (FCED) and to compare the endothelial properties of FCED, first-degree relatives, and controls. METHODS: Blood samples were collected from FCEDs to determine TNR number. The FCED patients, first-degree relatives, and controls were examined with specular microscopy for central corneal thickness (CCT), endothelial cell density (ECD), pleomorphism and polymegatism, and with corneal topography for specific indicators such as (i) displacement of thinnest point of cornea, (ii) loss of isopachs, (iii) focal posterior surface depression towards anterior chamber. RESULTS: This study included 92 patients with FCED, 92 first-degree relatives, and 96 controls. CCT was thickest in FCEDs (558.0 µm) (p < 0.05) while there was no difference between relatives (533.0 µm) and controls (530.4 µm) (p = 0.845). ECD was decreased in both FCED (2069.2 mm2) and relatives (2171.4 mm2) than controls (2822.9 mm2) (p < 0.05 in both). The presence of pleomorphism and polymegatism was significant in patients with FCED (93.4% and 93.4%, respectively), relatives (86.9% and 86.04%, respectively), and controls (8.33% and 1.04%, respectively) (p < 0.05). Specific topographic indicators differed among the groups (p < 0.05). The mean repeat number of the FCED patients was 17.48 ± 4.54 (12-25) times. The TNR number of FCED cases correlated with the relative CCT (p < 0.05, R = 0.615) and cell density (p = 0.009, R = -0.499). CONCLUSIONS: A strong association between the corneal endothelium in relatives and TNR number of FCEDs was defined. Relatives tended to have fewer corneal endothelial cells, even though they did not have clinical findings.

ß-catenin/TCF4-induced SCUBE3 upregulation promotes ovarian cancer development via HIF-1 signaling pathway.

The precise involvement and mechanistic role of the signal peptide-CUB-EGF-like domain-containing protein 3 (SCUBE3) in ovarian cancer (OV) remain poorly understood. Here, leveraging comprehensive data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, we unveil the selective overexpression of SCUBE3 in ovarian cancer tissues and cells. Intriguingly, elevated SCUBE3 expression levels correlate with an unfavorable prognosis in patients. Through meticulous manipulation of SCUBE3 expression, we elucidate its consequential impact on in vitro proliferation and invasion of ovarian cancer cells, as well as in vivo tumor growth in mice. Our multifaceted investigations, encompassing luciferase reporter assays, chromatin immunoprecipitation (ChIP) experiments, and mining of public databases, successfully identify SCUBE3 as a direct downstream target gene of TCF4-a pivotal positive regulator within the ß-catenin/TCF4 complex. Furthermore, utilizing a recessive mutant mouse line (kta41) harboring a functionally impaired point mutation at position 882 in the SCUBE3 gene, we uncover SCUBE3's involvement in the intricate regulation of angiogenesis and epithelial-mesenchymal transition (EMT). Strikingly, Spearman correlation coefficient analysis unveils a close association between SCUBE3 and HIF1A in OV, with SCUBE3 exerting tight control over HIF1A mRNA expression. Moreover, functional inhibition of HIF1A significantly impedes the pro-proliferative and invasive capabilities of SCUBE3-overexpressing ovarian cancer cells. Collectively, our findings underscore the pivotal role of SCUBE3 in driving ovarian cancer progression, shedding light on its intricate molecular mechanisms and establishing it as a potential therapeutic target for this devastating disease.

Role of maternally expressed 8 small nucleolar RNA (MEG8) in osteogenic differentiation of periodontal ligament stem cells.

OBJECTIVES: Periodontal ligament stem cells (PDLSCs) are essential for the treatment of bone diseases because of its great potential to differentiate into osteoblasts. Remarkably, increasing long-non-coding RNAs (lncRNAs) have been reported to be involved in the osteogenic differentiation of PDLSCs. Maternally expressed 8, small nucleolar RNA host gene (MEG8) is implicated in multiple diseases. This study intended to unearth the potential role of MEG8 and unveil the mechanism in PDLSCs undergoing osteoblastic differentiation. MATERIALS AND METHODS: MEG8 expression was measured by quantitative real-time PCR (RT-qPCR) during osteogenic differentiation of PDLSCs into bone cells. Functional assays were used to uncover the biological function of MEG8. Besides, RNA pulldown, RNA-binding protein immunoprecipitation (RIP), and luciferase reporter assays were used to explore the molecular mechanism of MEG8. RESULTS: MEG8 was apparently overexpressed in osteogenically differentiated PDLSCs. Moreover, MEG8 deficiency suppressed the osteoblastic differentiation of PDLSCs. Furthermore, MEG8 modulated the expression of transcription factor 4 (TCF4) by scavenging microRNA-495-3p (miR-495-3p) and microRNA-485-3p (miR-485-3p) through the competing endogenous RNA (ceRNA) mechanism, further stimulating the Wnt/ß-catenin pathway. CONCLUSION: MEG8 stimulates the capacity of PDLSCs for osteogenic differentiation through a ceRNA mode.

In vitro study of miRNA-369-3p targeting TCF4 regulating the malignant biological behavior of colon cancer cells.

Background: Colorectal carcinoma (CRC) is a common malignant tumor of the digestive tract. It is characterized by a high degree of malignancy, early metastasis and poor prognosis. Studies have shown the effect of miR-369-3p on the biological function of a variety of tumors. However, the mechanism by which miR-369-3p and its potential target genes participate in the pathogenesis of CRC has not been elucidated. This study aims to study the relationship between miR-369-3p and transcription factor 4 (TCF4), to reveal the mechanism of the occurrence and development of CRC, and to provide a promising target for the treatment of CRC. Methods: Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the miR-369-3p levels in CRC tissues and cells. miR-369-3p mimics and/or TCF4 overexpression vectors were transfected into SW480 cells. The expression of miR-369-3p and TCF4 mRNA was detected using RT-qPCR. Bioinformatics analysis predicted the binding site of miR-369-3p to the TCF4 3'UTR, and the targeting relationship was verified by a dual luciferase reporter gene assay. Cell proliferation and invasion were investigated by labeled immunofluorescence assay using BrdU antibody and Transwell assay. The oxidative stress ability of cells was determined by commercial kits. The levels of proteins related to cell proliferation and invasion were measured by western blotting. Results: The level of miR-369-3p was significantly down-regulated in CRC tissues and cell lines, especially in SW480 cells (P<0.05). The expression of TCF4 was negatively correlated with that of miR-369-3p. High levels of miR-369-3p targeting TCF4 suppressed cell proliferation and downregulated the protein expression of Ki67 and PCNA (P<0.05). Overexpressed miR-369-3p binding TCF4 inhibited cell invasion and decreased the protein levels of vascular endothelial growth factor (VEGF) and E-cadherin (P<0.05). Furthermore, upregulation of miR-369-3p increased the activity of superoxide dismutase (SOD) while decreasing the content of malondialdehyde (MDA) and activity of lactate dehydrogenase (LDH) by blocking the expression of TCF4 (P<0.05). Conclusions: MiR-369-3p inhibits the proliferation, invasion and oxidative stress of CRC cells by targeting TCF4, thus defining miR-369-3p as a potential target for the diagnosis and treatment of CRC.

Transcription Factor Id1 Plays an Essential Role in Th9 Cell Differentiation by Inhibiting Tcf3 and Tcf4.

T helper type 9 (Th9) cells play important roles in immune responses by producing interleukin-9 (IL-9). Several transcription factors are responsible for Th9 cell differentiation; however, transcriptional regulation of Th9 cells is not fully understood. Here, it is shown that Id1 is an essential transcriptional regulator of Th9 cell differentiation. Id1 is induced by IL-4 and TGF-ß. Id1-deficient naïve CD4 T cells fail to differentiate into Th9 cells, and overexpression of Id1 induce expression of IL-9. Mass spectrometry analysis reveals that Id1 interacts with Tcf3 and Tcf4 in Th9 cells. In addition, RNA-sequencing, chromatin immunoprecipitation, and transient reporter assay reveal that Tcf3 and Tcf4 bind to the promoter region of the Il9 gene to suppress its expression, and that Id1 inhibits their function, leading to Th9 differentiation. Finally, Id1-deficient Th9 cells ameliorate airway inflammation in an animal model of asthma. Thus, Id1 is a transcription factor that plays an essential role in Th9 cell differentiation by inhibiting Tcf3 and Tcf4.

USP10 promotes the progression of triple-negative breast cancer by enhancing the stability of TCF4 protein.

Investigating the role of ubiquitin-specific peptidase 10 (USP10) in triple-negative breast cancer (TNBC). Analyzed USP10 expression levels in tumors using public databases. Detected USP10 mRNA and protein levels in cell lines. Examined USP10 expression in tumor tissues from breast cancer patients. Conducted USP10 knockdown experiments and analyzed changes in cell proliferation and metastasis. Confirmed protein-protein interactions with USP10 through mass spectrometry, Co-IP, and fluorescence experiments. Assessed impact of USP10 on transcription factor 4 (TCF4) ubiquitination and validated TCF4's influence on TNBC cells. We initially identified a pronounced overexpression of USP10 across multiple tumor types, including TNBC. Subsequently, we observed a conspicuous upregulation of USP10 expression levels in breast cancer cell lines compared to normal breast epithelial cells. However, upon subsequent depletion of USP10 within cellular contexts, we noted a substantial attenuation of malignant proliferation and metastatic potential in TNBC cells. In subsequent experimental analyses, we elucidated the physical interaction between USP10 and the transcription factor TCF4, whereby USP10 facilitated the deubiquitination modification of TCF4, consequently promoting its protein stability and contributing to the initiation and progression of TNBC. Collectively, this study demonstrates that USP10 facilitated the deubiquitination modification of TCF4, consequently promoting its protein stability and contributing to the initiation and progression of TNBC.

18q21.1q21.32 Deletion in a Patient With Juvenile Cerebral Infarction.

The chromosome 18q deletion syndrome is a well-recognized chromosomal aberration characterized by intellectual disability, facial dysmorphism, short stature, microcephaly, cardiac anomalies, such as atrial and ventricular septal defect, and hypotonia; however, the phenotype is highly variable depending on the combination of genes within the chromosomal aberration regions. Thus far, no association was found between 18q deletion and cerebral infarction. Herein, we report a case of 18q deletion syndrome that caused juvenile cerebral infarction. A 32-year-old woman with an intellectual disability and facial dysmorphism presented with sudden-onset left-sided weakness. Brain magnetic resonance imaging revealed a striatocapsular infarction. Abnormalities in thrombotic profiles and embolic sources could not be identified. Microarray-based comparative genomic hybridization analysis detected a microdeletion in chromosome 18 encompassing the cytoregion 18q21.1q21.32. The deletion region contains the TCF4 and SMAD4 genes, whose haploinsufficiency causes the causative genes of Pitt-Hopkins syndrome (PTHS) and juvenile polyposis/hereditary hemorrhagic telangiectasia (JPHT or JPHHT), respectively. The patient's facial features were characteristic of PTHS, including a broad, beaked nasal bridge and a wide mouth with a bow-shaped upper lip. On the contrary, the patient did not show breathing abnormalities, which is one of the hallmarks of PTHS. We could not elucidate the relationship between cerebral infarction and genes included in the deleted region of 18q. However, if patients with chromosomal aberrations have cerebral infarctions, investigating the genes included within the chromosomal aberration regions may increase our knowledge of the genes involved in juvenile cerebral infarction.