Anlotinib Inhibits Ovarian Cancer and Enhances Cisplatinum Sensitivity via Suppressing NOTCH2 Expression and Stemness.

BACKGROUND/AIM: The prognosis of ovarian cancer (OC) patients is especially poor for patients with chemotherapy resistance. Anlotinib, a novel multi-targeted tyrosine kinase inhibitor, has shown encouraging clinical efficacy in several tumor types. The aim of the present study was to examine the inhibitory efficacy and mechanism of anlotinib on the proliferation and chemosensitivity of OC cells. MATERIALS AND METHODS: The inhibitory effects of Anlotinib on SKOV3 and OVCAR3 OC cells were examined using CCK-8 cell-viability, colony-formation, flow-cytometry, transwell-migration and sphere-formation assays. A xenograft mouse model was used for in vivo studies. RT-qPCR and western blotting were used to detect gene expression. RESULTS: Molecular targets of anlotinib were elevated in OC patient tumors. Anlotinib significantly inhibited ovarian cancer cell proliferation and migration in vitro. Anlotinib enhanced the sensitivity of ovarian cancer cells to cisplatinum both in vitro and in vivo. Anlotinib suppressed sphere formation and the stemness phenotype of OC cells by inhibiting NOTCH2 expression. CONCLUSION: Anlotinib inhibits ovarian cancer and enhances cisplatinum sensitivity, suggesting its future clinical promise.

Glycoprotein hormone subunit alpha 2 (GPHA2): A pituitary stem cell-expressed gene associated with NOTCH2 signaling.

NOTCH2 is expressed in pituitary stem cells and is necessary for stem cell maintenance, proliferation, and differentiation. However, the pathways NOTCH2 engages to affect pituitary development remain unclear. In this study, we hypothesized that glycoprotein hormone subunit A2 (GPHA2), a corneal stem cell factor and ligand for the thyroid stimulating hormone receptor (TSHR), is downstream of NOTCH2 signaling. We found Gpha2 is expressed in quiescent pituitary stem cells by RNAscope in situ hybridization and scRNA seq. In Notch2 conditional knockout pituitaries, Gpha2 mRNA is reduced compared with control littermates. We then investigated the possible functions of GPHA2. Pituitaries treated with a GPHA2 peptide do not have a change in proliferation. However, in dissociated adult pituitary cells, GPHA2 increased pCREB expression and this induction was reversed by co-treatment with a TSHR inhibitor. These data suggest GPHA2 is a NOTCH2 related stem cell factor that activates TSHR signaling, potentially impacting pituitary development.

NOTCH2 promotes osteoclast maturation and metabolism and modulates the transcriptome profile during osteoclastogenesis.

Notch signaling plays a key regulatory role in bone remodeling and NOTCH2 enhances osteoclastogenesis, an effect that is mostly mediated by its target gene Hes1. In the present study, we explored mechanisms responsible for the enhanced osteoclastogenesis in bone marrow-derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice. Notch2tm1.1Ecan mice are osteopenic and have enhanced osteoclastogenesis. Bulk RNA-Seq and gene set enrichment analysis of Notch2tm1.1Ecan BMMs cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand revealed enrichment of genes associated with enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis. These pathways were not enhanced in the context of a Hes1 inactivation. Analysis of single cell RNA-Seq data of pooled control and Notch2tm1.1Ecan BMMs treated with M-CSF or M-CSF and receptor activator of NF-κB ligand for 3 days identified 11 well-defined cellular clusters. Pseudotime trajectory analysis indicated a trajectory of clusters expressing genes associated with osteoclast progenitors, osteoclast precursors, and mature cells. There were an increased number of cells expressing gene markers associated with the osteoclast and with an unknown, albeit related, cluster in Notch2tm1.1Ecan than in control BMMs as well as enhanced expression of genes associated with osteoclast progenitors and precursors in Notch2tm1.1Ecan cells. In conclusion, BMM cultures display cellular heterogeneity, and NOTCH2 enhances osteoclastogenesis, increases mitochondrial and metabolic activity of osteoclasts, and affects cell cluster allocation in BMMs.

[Prognostic model for assessing the human glioma cell malignancy grade based on MDM2, MELK, SOX2, CDK4, DR5 and OCT4 gene expression]. / Prognosticheskaya model' dlya otsenki stepeni zlokachestvennosti kul'tury kletok gliomy cheloveka na osnovanii issledovaniya ekspressii paneli genov MDM2, MELK, SOX2, CDK4, DR5 i OCT4.

Glioma cell cultures are used in basic researches of tumor processes, personalized medicine for selecting treatment regimens depending on individual characteristics of patients and pharmacology for assessing the effectiveness of chemotherapy. Suppression of glioma culture growth without reduction of malignancy grade is common. Drug cancellation may be followed by substitution of precursor cells by more malignant clones. Therefore, analysis of culture cell malignancy grade is important. In the future, intraoperative analysis of glioma cell malignancy grade can be used to select individual therapy. OBJECTIVE: We analyzed the relationship between expression of marker genes TUBB3, CD133, CDK4, CDK6, CIRBP, DR4, DR5, EGFR, FGFR, FSHR, GDNF, GFAP, L1CAM, LEF1, MAP2, MDM2, MELK, NANOG, NOTCH2, OCT4, OLIG2, PDGFRA, PDGFA, PDGFB and SOX2 and glioma cell malignancy grade, as well as created appropriate prognostic model. MATERIAL AND METHODS: We analyzed expression of 25 marker genes in 22 samples of human glioma cultures using quantitative real-time PCR. Statistical analysis was performed using the IBM SPSS Statistics 26.0 software. We used the Kolmogorov-Smirnov and Shapiro-Wilk tests to assess distribution normality. Nonparametric Jonckheere-Terpstra and Spearman tests were applied. RESULTS: We obtained a prognostic model for assessing the grade III and IV glioma cell malignancy based on expression of marker genes MDM2, MELK, SOX2, CDK4, DR5 and OCT4. Predictive accuracy was 83% (Akaike information criterion -55.125).

Pan-cancer tRNA-derived fragment CAT1 coordinates RBPMS to stabilize NOTCH2 mRNA to promote tumorigenesis.

Transfer RNA-derived fragments (tRFs) are a class of small non-coding regulatory RNAs that are involved in the pathophysiology of many diseases. However, the role of tRFs in cancer progression remains largely elusive. Here, we demonstrate that a pan-cancer 3'-tRF, CAT1 (cancer associated tRF 1), is ubiquitously upregulated in tumors and associated with poor prognosis of a variety of cancers, including lung cancer. The upregulated CAT1 in cancer cells binds to RNA-binding protein with multiple splicing (RBPMS) and displaces NOTCH2 association from RBPMS, thereby inhibiting the subsequent CCR4-NOT deadenylation-complex-mediated NOTCH2 mRNA decay. The CAT1-enhanced NOTCH2 expression promotes lung cancer cell proliferation and metastasis in vitro and in vivo. In addition, plasma CAT1 levels are substantially increased in patients with lung cancer compared to non-cancer control subjects. Our findings reveal an intrinsic connection between cancer-specific upregulation of CAT1 and cancer progression, show the regulation of NOTCH signaling in cancer by a 3'-tRF, and highlight its great clinical potential.

NOTCH2 sensitizes the chondrocyte to the inflammatory response of tumor necrosis factor α.

Notch regulates the immune and inflammatory response and has been associated with the pathogenesis of osteoarthritis in humans and preclinical models of the disease. Notch2tm1.1Ecan mice harbor a NOTCH2 gain-of-function and are sensitized to osteoarthritis, but the mechanisms have not been explored. We examined the effects of tumor necrosis factor α (TNFα) in chondrocytes from Notch2tm1.1Ecan mice and found that NOTCH2 enhanced the effect of TNFα on Il6 and Il1b expression. Similar results were obtained in cells from a conditional model of NOTCH2 gain-of-function, Notch22.1Ecan mice, and following the expression of the NOTCH2 intracellular domain in vitro. Recombination signal-binding protein for immunoglobulin Kappa J region partners with the NOTCH2 intracellular domain to activate transcription; in the absence of Notch signaling it inhibits transcription, and Rbpj inactivation in chondrocytes resulted in Il6 induction. Although TNFα induced IL6 to a greater extent in the context of NOTCH2 activation, there was a concomitant inhibition of Notch target genes Hes1, Hey1, Hey2, and Heyl. Electrophoretic mobility shift assay demonstrated displacement of recombination signal-binding protein for immunoglobulin Kappa J region from DNA binding sites by TNFα explaining the increased Il6 expression and the concomitant decrease in Notch target genes. NOTCH2 enhanced the effect of TNFα on NF-κB signaling, and RNA-Seq revealed increased expression of pathways associated with inflammation and the phagosome in NOTCH2 overexpressing cells in the absence and presence of TNFα. Collectively, NOTCH2 has important interactions with TNFα resulting in the enhanced expression of Il6 and inflammatory pathways in chondrocytes.

Specific knockout of Notch2 in Treg cells significantly inhibits the growth and proliferation of head and neck squamous cell carcinoma in mice.

OBJECTIVE: To investigate the effect of Notch2 gene knockout in Treg cells on head and neck squamous cell carcinoma (HNSCC) in mice. METHODS: A mouse model of HNSCC was constructed. Flow cytometry and immunofluorescence were used to examine the numbers of related immune cells and programmed cell death in tumor cells in the spleen and tumor microenvironment of mice. Western blotting was used to measure the expression of related proteins in tumor tissues. RESULTS: The tumor volume of regulatory T (Treg) cell-specific Notch2-knockout mice (experimental group) was significantly smaller than that of control mice (control group) (P < 0.05). Compared with those in the control group, the number of Treg cells and the expression of Ki67 in Treg cells in the spleen and tumor tissue were significantly decreased in the experimental group, while the numbers of CD45+ hematopoietic cells, CD4+ T cells, CD8+ T cells, T helper 1 (Th1) cells, CD11b+ cells (macrophages), and CD11b+CD11c+ cells (dendritic cells) and the expression of Ki67 in CD4+ T cells and CD8+ T cells were significantly increased (P < 0.05). There was no significant difference in the number of Th2 cells between the two groups (P > 0.05). Immunofluorescence analysis showed that the numbers of CD4+ T cells and CD8+ T cells in the tumor tissue in the experimental group were significantly higher than those in the control group (P < 0.05). Compared with that in the control group, programmed cell death in the experimental group was significantly increased (P < 0.05). Moreover, the expression levels of NLRP3, Caspase-1 and GSDMD in the tumor tissues of the experimental group were higher than those in the control group (P < 0.01), while the expression levels of BCL2, Bax, ATG5, LC3 and p62 were not significantly different (P > 0.05). CONCLUSIONS: Specific knockout of the Notch2 gene in Treg cells significantly decreases the function of Treg cells, inhibits the growth of HNSCC and improves the immune microenvironment in mice, thus effectively treating HNSCC.

NOTCH2NLC GGC repeat expansion causes retinal pathology with intranuclear inclusions throughout the retina and causes visual impairment.

The retinal pathology of genetically confirmed neuronal intranuclear inclusion disease (NIID) is yet unknown. We report the ocular findings in four NIID patients with NOTCH2NLC GGC repeat expansion to investigate the pathology of retinopathy. All four NIID patients were diagnosed by skin biopsy and NOTCH2NLC GGC repeat analysis. Ocular findings in patients with NIID were studied using fundus photographs, optical coherence tomographic images (OCT), and full-field electroretinograms (ERGs). The histopathology of the retina was studied on autopsy samples from two cases with immunohistochemistry. All patients had an expansion of the GGC repeat (87-134 repeats) in the NOTCH2NLC. Two patients were legally blind and had been diagnosed with retinitis pigmentosa prior to the diagnosis of NIID and assessed with whole exome sequencing to rule out comorbidity with other retinal diseases. Fundus photographs around the posterior pole showed chorioretinal atrophy in the peripapillary regions. OCT showed thinning of the retina. ERGs showed various abnormalities in cases. The histopathology of autopsy samples showed diffusely scattered intranuclear inclusions throughout the retina from the retinal pigment epithelium to the ganglion cell layer, and optic nerve glial cells. And severe gliosis was observed in retina and optic nerve. The NOTCH2NLC GGC repeat expansion causes numerous intranuclear inclusions in the retina and optic nerve cells and gliosis. Visual dysfunction could be the first sign of NIID. We should consider NIID as one of the causes of retinal dystrophy and investigate the GGC repeat expansion in NOTCH2NLC.

DLBCL-associated NOTCH2 mutations escape ubiquitin-dependent degradation and promote chemoresistance.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Up to 40% of patients with DLBCL display refractory disease or relapse after standard chemotherapy treatment (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone [R-CHOP]), leading to significant morbidity and mortality. The molecular mechanisms of chemoresistance in DLBCL remain incompletely understood. Using a cullin-really interesting new gene (RING) ligase-based CRISPR-Cas9 library, we identify that inactivation of the E3 ubiquitin ligase KLHL6 promotes DLBCL chemoresistance. Furthermore, proteomic approaches helped identify KLHL6 as a novel master regulator of plasma membrane-associated NOTCH2 via proteasome-dependent degradation. In CHOP-resistant DLBCL tumors, mutations of NOTCH2 result in a protein that escapes the mechanism of ubiquitin-dependent proteolysis, leading to protein stabilization and activation of the oncogenic RAS signaling pathway. Targeting CHOP-resistant DLBCL tumors with the phase 3 clinical trial molecules nirogacestat, a selective γ-secretase inhibitor, and ipatasertib, a pan-AKT inhibitor, synergistically promotes DLBCL destruction. These findings establish the rationale for therapeutic strategies aimed at targeting the oncogenic pathway activated in KLHL6- or NOTCH2-mutated DLBCL.

Clinical significance of Notch pathway-associated microRNA-107 in pancreatic ductal adenocarcinoma.

Background & aim: MicroRNAs associated with the Notch pathway play a critical role in the progression of pancreatic carcinoma. Our aim was to study the clinical significance of miR-107 and NOTCH2 in pancreatic ductal adenocarcinoma (PDAC). Methods: The circulating miR-107 levels in PDAC and controls were determined by qPCR. NOTCH2 protein (target) expression in tissue of PDAC, periampullary carcinoma, chronic pancreatitis and normal pancreatic tissue was assessed by immunohistochemistry. Results: The circulating miR-107 levels were found to be significantly reduced in PDAC as compared with controls. Additionally, NOTCH2 protein expression was higher in PDAC tissue as compared with controls and was clinically associated with metastasis. Conclusion: Our findings demonstrate the utility of circulating miR-107 as a potential differentiating marker in PDAC.

NOTCH2 related disorders: Description and review of the fetal presentation.

Signs of skeletal dysplasias are relatively common in fetuses with abnormal ultrasound (US) findings. The diversity of congenital skeletal disorders, the possibility of late-onset severe phenotypes and overlapping syndromes can be a challenge in the way of diagnosis, even if prenatal high-throuput sequencing allows for a better diagnosis, prognosis and genetic counseling. Hajdu-Cheney spectrum pathologies are rarely described in prenatal, and the signs associated remain poorly known, and do not include specific postnatal signs as acro-osteolysis and premature osteoporosis. We hereby report a couple for whom a medical termination of pregnancy was performed because a severe polymalformative syndrome associating severely short limbs with bowed long bones, severe cardiopathy, hyperechogenic kidneys and dysmorphism. After fetopathological and radiological examinations, Exome Sequencing (ES) was performed and revealed a de novo truncating mutation in the last exon of NOTCH2, responsible for Hajdu-Cheney or Serpentine Fibula Polycystic Kidney syndromes.

[DTX2 overexpression promotes migration and invasion of colorectal cancer cells through the Notch2/Akt axis].

OBJECTIVE: To investigate the effect of changes in DTX2 expression level on migration and invasion of colorectal cancer (CRC) cells and explore the mechanism. METHODS: Two CRC cell lines SW620 and LoVo were transfected with a specific shRNA targeting DTX2 (DTX2-shRNA) or a DTX2-overexpressing plasmid (pcDNA-DTX2), and the transfection efficiency was evaluated with RT-qPCR and Western blotting. Scratch and Transwell assays were used to assess the changes in migration and invasion ability of the transfected cells, and the cellular expression levels of Notch2, NICD, AKT, p-Akt and MMP-2/9 proteins were detected with Western blotting. The CRC cells were co-transfected with pcDNA-DTX2 and Notch2 siRNA to assess the effect of Notch2 knockdown on DTX2 overexpression-induced enhancement of cell migration and invasion. RESULTS: The expression levels of DTX2 at both the mRNA and protein levels were significantly decreased in CRC cells transfected with DTX2- shRNA (P < 0.01) and increased in cells transfected with pcDNA-DTX2 (P < 0.01). Scratch and Transwell assays showed that the migration and invasion abilities of CRC cells were significantly lowered following DTX2 knockdown (P < 0.01) and were enhanced in cells with DTX2 overexpression (P < 0.01). The expression levels of Notch2, NICD, p-Akt and MMP-2 proteins decreased significantly in CRC cells with DTX2 knockdown (P < 0.05) and increased obviously in DTX2-overexpressing cells (P < 0.05). In both of the two CRC cell lines, transfection with Notch2 siRNA obviously reversed the effect of DTX2 overexpression in promoting cell migration and invasion (P < 0.01) and expressions of the related proteins. CONCLUSION: DTX2 overexpression promotes migration and invasion of CRC cells through the Notch2/Akt axis, suggesting the potential of DTX2 as a new biological indicator of CRC.

Coordinated modification in expression levels of HSPA1A/B, DGKH, and NOTCH2 in Parkinson's patients' blood and substantia nigra as a diagnostic sign: the transcriptomes' relationship.

BACKGROUND: Diagnosis of Parkinson's disease (PD) is associated with a vast number of challenges. This study aimed to assess the overlap of PD patients' transcriptomes in the substantia nigra (SN) with peripheral blood mononuclear cells (PBMCs) to discover potential biomarkers for diagnosis. METHODS: GEO data were used to select genes with significant changes in expression level in the SN region and eligible studies. Also, transcriptome data related to blood of PD patients with other neurodegenerative diseases (ND) was considered. Differential expression genes between PD and control were evaluated in the SN and blood, and RT-qPCR was applied to validate the findings. RESULTS: At the expression level, no significant similarity in long non-coding RNA was found between the patients' SN and blood. While in silico results revealed 16 common mRNAs in SN and blood with significant expression levels. Among all overexpressed mRNAs, HSPA1A/B expression level had the highest expression difference between control and PD samples. Moreover, DGKH had the highest score of down-regulated genes in both blood and SN. The NOTCH pathway had the highest score pathway among up-regulated pathways, and the expression levels of NOTCH2, H4C8, and H2BC21 associated with this pathway had the most ability to separate the control and PD populations. Furthermore, RT-qPCR results revealed that HSPA1A/B, NOTCH2, and H4C8 were overexpressed in PD PBMCs, while DGKH expression levels were lower compared to controls. CONCLUSION: Our findings indicate that expression levels of HSPA1A/B, DGKH, and NOTCH2 could be applied as candidate biomarkers to diagnose PD patients in the SN region and PBMCs.

Long Non-Coding RNA LINC00052 Targets miR-548p/Notch2/Pyk2 to Modulate Tumor Budding and Metastasis of Human Breast Cancer.

Abnormal expression of long non-coding RNAs (lncRNAs) is involved in many pathological processes of cancers. However, the role of lncRNA LINC00052 in breast cancer progression is still unclear. Here, LINC00052 expression was detected by in situ hybridization and quantitative real-time PCR assays. Cell Counting Kit-8, wound healing, and transwell assays were used to investigate changes in the proliferation, migration, and invasion of breast cancer cells. MiR-548p was found associated with LINC00052 or Notch2 by RNA pull-down, dual-luciferase reporter, and qRT-PCR assays. The effect of LINC00052 on lung metastasis was explored through in vivo experiments. High LINC00052 expression was observed in breast cancer tissues and cells. LINC00052 silencing inhibited the proliferation, migration, and invasion of MCF7 cells, and LINC00052 overexpression produced the opposite results. MiR-548p, a target gene of LINC00052, partially rescued the effects of LINC00052 on proliferation, migration, and invasion of MCF7. Notch2 was the target of miR-548p and LINC00052 could promote Notch2 expression. Moreover, the phosphorylation of proline-rich tyrosine kinase 2 (Pyk2), a downstream factor of Notch2, was increased by LINC00052, and a Pyk2 mutant could inhibit the cell migration and invasion induced by LINC00052 overexpression in MDA-MB-468 cells, which was similar to the function of the miR-548p mimic. We further demonstrated that LINC00052 exacerbated the metastases of breast cancer cells in vivo. Our research demonstrated that LINC00052 is highly expressed in breast cancer and promotes breast cancer proliferation, migration, and invasion via the miR-548p/Notch2/Pyk2 axis. LINC00052 could serve as a potential therapeutic target for breast cancer.

TP53, NOTCH2, and STK11 Mutations in a Rare Tumor of non-Small Cell Lung Carcinoma with Diffuse Coexpression of TTF1 and p40 in the Same Tumor Cells.

Introduction. Five cases of non-small cell lung carcinoma (NSCLC) with diffuse coexpression of TTF1 and p40 in the same tumor cells (hereafter referred to as TTF1/P40-NSCLC) have been reported since 2015. It was considered a new entity of NSCLC occurred in aged male smokers with poorly differentiated carcinomas and a similar molecular signature harboring a TP53 mutation. Methods. We report an extremely rare tumor of TTF1/P40-NSCLC. Morphological observation and immunohistochemical examination were performed, clinical and molecular features were summarized, and a review of the relevant literature was provided. Results. The tumor showed a solid growth pattern with patchy necrosis, and glandular and squamous pattern were not obvious. The tumor cells proliferated within the bronchial epithelium. Spreading through air spaces of tumor cells were observed. A peculiar immunohistochemical phenotype of diffuse and strong positivity for TTF1 (8G7G3/1) and p40 in the same tumor cells was detected. Additionally, the tumor cells were positive for KRT7 and KRT20, while negative for PD-L1 (22C3). Negative P53 (null) Immunohistochemistry (IHC) staining indicated mutational status and the Ki67 index was 80%. Molecular investigation was performed using whole exome sequencing, and TP53, NOTCH2, and STK11 mutations were detected. The patient remained alive over a follow-up period of 22 months without tumor recurrence or metastasis. Conclusions. We describe an unusual tumor of TTF1/P40-NSCLC harboring TP53, NOTCH2 and STK11 mutations. These gene mutations may be helpful in providing additional therapeutic possibilities. Our report offers further insight into this rare tumor.

Hajdu-Cheney syndrome with atypical cardiovascular abnormalities.

Hajdu-Cheney syndrome is an ultra-rare autosomal dominant disorder caused by a heterozygous variant in NOTCH2 gene. Characteristic features include osteolysis, distinct facial appearance, skull deformity, joint laxity, osteoporosis, and short stature. Associated abnormalities are congenital heart disease, congenital defects of the kidney, and neurological problems. Here, we present the first reported case of an African child with a variant in NOTCH2 gene and features of Hajdu-Cheney syndrome in whom we detected a congenital heart defect that has not been previously reported in association with the syndrome. To appropriately characterize this disease and document correct proportion of cardiovascular malformation associations, echocardiography is recommended for all cases of Hajdu Cheney syndrome.

PIK3R3 Missense and NOTCH2 Synonymous Single Nucleotide Polymorphisms Are Associated with Liver Cancer.

BACKGROUND: Single nucleotide polymorphism (SNP) of candidate genes also affects the occurrence and prognosis of liver cancer. We mainly explored the effects of PIK3R3 and NOTCH2 polymorphisms on liver cancer risk among Chinese people. METHODS: Four SNPs (rs785468, rs785467, rs3795666, and rs17024525 in PIK3R3 and NOTCH2) from 709 liver cancer patients and 700 healthy controls were genotyped using the Agena MassARRAY system. The correlation between SNPs and liver cancer risk was evaluated using logistic regression analysis. The SNP-SNP interactions were conducted by the multifactor dimensionality reduction method. RESULTS: The results revealed that PIK3R3-rs785467 reduced the likelihood of liver cancer among Chinese Han people (p < 0.05). In addition, PIK3R3-rs785467 decreased the susceptibility to liver cancer in different populations (females, non-smokers, and age >55 years, p < 0.05). NOTCH2-rs3795666 reduced the susceptibility to liver cancer among males, drinkers, and patients aged >55 years (p < 0.05). CONCLUSIONS: Our results demonstrate that PIK3R3-rs785476 and NOTCH2-rs3795666 polymorphisms are responsible for decreasing the susceptibility of liver cancer development in the Chinese Han population.

Bone marrow mesenchymal stem cell-derived exosomes shuttling miR-150-5p alleviates mechanical allodynia in rats by targeting NOTCH2 in microglia.

BACKGROUND: This study probes into the function and mechanism of bone marrow mesenchymal stem cell (BMSC)-derived exosomes loaded with miR-150-5p in mechanical allodynia. METHODS: BMSCs were infected with miR-150-5p inhibition lentiviruses to obtain exosomes with low miR-150-5p expression. A L5 spinal nerve ligation (SNL) model was established in rats where exosomes, NOTCH2 overexpression/inhibition plasmids, or microglial cells were intrathecally administered. Hind paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) of rats were measured. TUNEL staining was used to measure the apoptotic rate in rat spinal dorsal horn (SDH), ELISA to evaluate pro-inflammatory factor levels, and RT-qPCR, western blotting, and immunohistochemistry to detect miR-150-5p and NOTCH2 expression. Immunofluorescence was used for localizing exosomes and NOTCH2 and detecting the expression of OX42, a maker for microglia. Dual luciferase reporter and RNA pull down assays were performed to validate the putative binding between miR-150-5p and NOTCH2. RESULTS: NOTCH2 expressed at a high level and miR-150-5p was downregulated in SDH of SNL rats. Exosomes injected were localized in rat SDH. BMSC-exosomes or NOTCH2 downregulation increased PWT and PWL of SNL rats and reduced apoptosis and inflammation in SDH. In contrast, NOTCH2 overexpression aggravated mechanical allodynia and SDH injury. Moreover, inhibiting miR-150-5p in BMSC-exosomes offset the therapeutic effects of BMSC-exosomes. Microglia activation induced mechanical allodynia in wild rats, while intrathecal injection of microglial cells incubated with BMSC-exosomes showed alleviated mechanical allodynia in SNL rats. NOTCH2 was targeted by miR-150-5p. CONCLUSION: BMSC-derived exosomal miR-150-5p alleviates mechanical allodynia by targeting NOTCH2 in microglial cells.

Pathogenic Novel Heterozygous Variant c.1076c>T p. (Ser359Phe) chr1: 120512166 in NOTCH2 Gene, Type 2 Alagille Syndrome Causing Neonatal Cholestasis: A Case Report.

BACKGROUND Alagille syndrome (ALGS) is a multisystem hereditary illness with a dominant pattern and partial penetrance. Multiple organ abnormalities can be caused by mutations in the Jagged canonical Notch ligand 1 (JAG1) gene. Notch receptor 2 (NOTCH2) gene mutations are also uncommon. ALGS is also characterized by deformed or narrowed bile ducts and is notoriously difficult to diagnose due to the wide range of symptoms and absence of unambiguous genotype-phenotype connections. Little is known about ALGS patients who have NOTCH2 mutations. We present a patient who developed progressive liver failure due to a unique pathogenic heterozygous variation of the NOTCH2 gene, c.1076c>T p. (Ser359Phe) chr1: 120512166, resulting in type 2 ALGS. CASE REPORT A Saudi Arabian newborn with bilateral hazy eyes, ectropion, dry ichthyic skin, normal male genitalia, and bilateral undescended testes was born at 31 weeks. Previous miscarriages, pregnancy-induced maternal cholestasis, fatty liver, or neonatal jaundice were not reported in the family history. He had developed worsening cholestatic jaundice by the third week of hospitalization. The extensive work-up for metabolic, infectious, and other relevant etiologies was negative. Following gram-negative sepsis, he died of multiorgan failure. A NOTCH2 gene mutation explained the phenotypic difference in our situation. Another intriguing observation was the presence of ichthysis and craniosynostosis in ALGS with a NOTCH2 mutation. CONCLUSIONS Cholestasis in newborns can be difficult to diagnose. Next-generation sequencing detects 112 copy number variants in the cholestasis gene panel blood test. More research is needed to understand why NOTCH2 mutations are relatively rare in ALGS.