ID1high/activin Ahigh glioblastoma cells contribute to resistance to anti-angiogenesis therapy through malformed vasculature.

Although bevacizumab (BVZ), a representative drug for anti-angiogenesis therapy (AAT), is used as a first-line treatment for patients with glioblastoma (GBM), its efficacy is notably limited. Whereas several mechanisms have been proposed to explain the acquisition of AAT resistance, the specific underlying mechanisms have yet to be sufficiently ascertained. Here, we established that inhibitor of differentiation 1 (ID1)high/activin Ahigh glioblastoma cell confers resistance to BVZ. The bipotent effect of activin A during its active phase was demonstrated to reduce vasculature dependence in tumorigenesis. In response to a temporary exposure to activin A, this cytokine was found to induce endothelial-to-mesenchymal transition via the Smad3/Slug axis, whereas prolonged exposure led to endothelial apoptosis. ID1 tumors showing resistance to BVZ were established to be characterized by a hypovascular structure, hyperpermeability, and scattered hypoxic regions. Using a GBM mouse model, we demonstrated that AAT resistance can be overcome by administering therapy based on a combination of BVZ and SB431542, a Smad2/3 inhibitor, which contributed to enhancing survival. These findings offer valuable insights that could contribute to the development of new strategies for treating AAT-resistant GBM.

The efficacy of sorafenib against hepatocellular carcinoma is enhanced by 5-aza-mediated inhibition of ID1 promoter methylation.

Sorafenib resistance greatly restricts its clinical application in patients with hepatocellular carcinoma (HCC). Numerous studies have reported that ID1 exerts a crucial effect in cancer initiation and development. Our previous research revealed an inhibitory role of ID1 in sorafenib resistance. However, the upstream regulatory mechanism of ID1 expression is unclear. Here, we discovered that ID1 expression is negatively correlated with promoter methylation, which is regulated by DNMT3B. Knockdown of DNMT3B significantly inhibited ID1 methylation status and resulted in an increase of ID1 expression. The demethylating agent 5-aza-2'-deoxycytidine (5-aza) remarkably upregulated ID1 expression. The combination of 5-aza with sorafenib showed a synergistic effect on the inhibition of cell viability.

ID1 expressing macrophages support cancer cell stemness and limit CD8+ T cell infiltration in colorectal cancer.

Elimination of cancer stem cells (CSCs) and reinvigoration of antitumor immunity remain unmet challenges for cancer therapy. Tumor-associated macrophages (TAMs) constitute the prominant population of immune cells in tumor tissues, contributing to the formation of CSC niches and a suppressive immune microenvironment. Here, we report that high expression of inhibitor of differentiation 1 (ID1) in TAMs correlates with poor outcome in patients with colorectal cancer (CRC). ID1 expressing macrophages maintain cancer stemness and impede CD8+ T cell infiltration. Mechanistically, ID1 interacts with STAT1 to induce its cytoplasmic distribution and inhibits STAT1-mediated SerpinB2 and CCL4 transcription, two secretory factors responsible for cancer stemness inhibition and CD8+ T cell recruitment. Reducing ID1 expression ameliorates CRC progression and enhances tumor sensitivity to immunotherapy and chemotherapy. Collectively, our study highlights the pivotal role of ID1 in controlling the protumor phenotype of TAMs and paves the way for therapeutic targeting of ID1 in CRC.

Transcription factor 3 promotes migration and invasion potential and maintains cancer stemness by activating ID1 expression in esophageal squamous cell carcinoma.

Transcription factor 3 (TCF3) is a member of the basic Helix - Loop - Helix (bHLH) transcription factor (TF) family and is encoded by the TCF3 gene (also known as E2A). It has been shown that TCF3 functions as a key transcription factor in the pathogenesis of several human cancers and plays an important role in stem cell maintenance and carcinogenesis. However, the effect of TCF3 in the progression of esophageal squamous cell carcinoma (ESCC) is poorly known. In our study, TCF3 was found to express highly and correlated with cancer stage and prognosis. TCF3 was shown to promote ESCC invasion, migration, and drug resistance both from the results of in vivo and in vitro assays. Moreover, further studies suggested that TCF3 played these roles through transcriptionally regulating Inhibitor of DNA binding 1(ID1). Notably, we also found that TCF3 or ID1 was associated with ESCC stemness. Furthermore, TCF3 was correlated with the expression of cancer stemness markers CD44 and CD133. Therefore, maintaining cancer stemness might be the underlying mechanism that TCF3 transcriptionally regulated ID1 and further promoted ESCC progression and drug resistance.

Nicotinamide adenine dinucleotide kinase promotes lymph node metastasis of NSCLC via activating ID1 expression through BMP pathway.

Metastasis is a significant cause of high mortality in lung cancer. Lymph node (LN) metastasis is the most common metastatic pathway in non-small cell lung cancer and the most crucial factor affecting the prognosis of NSCLC. Nevertheless, the molecular mechanism underlying metastasis is unknown. We demonstrated that higher NADK expression suggests worsened survival prognosis, and NADK expression positively correlates with the lymph node metastasis rate and TNM and AJCC stages in NSCLC patients. Moreover, patients with LN metastasis show higher NADK expression than those without LN metastasis. NADK can promote NSCLC progression by enhancing the migration, invasion, lymph node metastasis and growth of NSCLC cells. Mechanistically, NADK inhibits the ubiquitination and degradation of BMPR1A by interacting with Smurf1, further activating the BMPs signalling pathway and promoting ID1 transcription. In conclusion, NADK may be a potential diagnostic indicator and a novel therapeutic target for metastatic NSCLC.

STAT3-Mediated Promoter-Enhancer Interaction Up-Regulates Inhibitor of DNA Binding 1 (ID1) to Promote Colon Cancer Progression.

BACKGROUND: High expression of inhibitor of DNA binding 1 (ID1) correlates with poor prognosis in colorectal cancer (CRC). Aberrant enhancer activation in regulating ID1 transcription is limited. METHODS: Immunohistochemistry (IHC), quantitative RT-PCR (RT-qPCR) and Western blotting (WB) were used to determine the expression of ID1. CRISPR-Cas9 was used to generate ID1 or enhancer E1 knockout cell lines. Dual-luciferase reporter assay, chromosome conformation capture assay and ChIP-qPCR were used to determine the active enhancers of ID1. Cell Counting Kit 8, colony-forming, transwell assays and tumorigenicity in nude mice were used to investigate the biological functions of ID1 and enhancer E1. RESULTS: Human CRC tissues and cell lines expressed a higher level of ID1 than normal controls. ID1 promoted CRC cell proliferation and colony formation. Enhancer E1 actively regulated ID1 promoter activity. Signal transducer and activator of transcription 3 (STAT3) bound to ID1 promoter and enhancer E1 to regulate their activity. The inhibitor of STAT3 Stattic attenuated ID1 promoter and enhancer E1 activity and the expression of ID1. Enhancer E1 knockout down-regulated ID1 expression level and cell proliferation in vitro and in vivo. CONCLUSIONS: Enhancer E1 is positively regulated by STAT3 and contributes to the regulation of ID1 to promote CRC cell progression and might be a potential target for anti-CRC drug studies.

Abnormal regulation of miR-29b-ID1 signaling is involved in the process of decitabine resistance in leukemia cells.

Decitabine (DAC) is an inhibitor of DNA methyltransferase used to treat leukemia, but primary or secondary resistance to DAC may develop during therapy. The mechanisms related to DAC resistance remain poorly understood. In this study, we find that miR-29b expression was decreased in various leukemia cell lines and AML patients and was associated with poor prognosis. In DAC-sensitive cells, miR-29b inhibited cell growth, promoted apoptosis, and increased the sensitivity to DAC. Similarly, it exerted anti-leukemic effects in DAC-resistant cells. When the miR-29b promoter in DAC-resistant cells was demethylated, its expression was not up-regulated. Furthermore, the expression of ID1, one of the target genes of miR-29b, was down-regulated in miR-29b transfected leukemic cells. ID1 promoted cell growth, inhibited cell apoptosis, and decreased DAC sensitivity in leukemic cells in vitro and in vivo. ID1 was down-regulated in DAC-sensitive cells treated with DAC, while it was up-regulated in DAC-resistant cells. Interestingly, the ID1 promoter region was completely unmethylated in both DAC-resistant cells and sensitive cells before DAC treatment. The growth inhibition, increased DAC sensitivity, and apoptosis induced by miR-29b can be eliminated by increasing ID1 expression. These results suggested that DAC regulates ID1 expression by acting on miR-29b. Abnormal ID1 expression of ID1 that is methylation independent and induced by miR-29b may be involved in the process of leukemia cells acquiring DAC resistance.

TWEAK Signaling-Induced ID1 Expression Drives Malignant Transformation of Hepatic Progenitor Cells During Hepatocarcinogenesis.

The malignant transformation of hepatic progenitor cells (HPCs) in the inflammatory microenvironment is the root cause of hepatocarcinogenesis. However, the potential molecular mechanisms are still elusive. The HPCs subgroup is identified by single-cell RNA (scRNA) sequencing and the phenotype of HPCs is investigated in the primary HCC model. Bulk RNA sequencing (RNA-seq) and proteomic analyses are also performed on HPC-derived organoids. It is found that tumors are formed from HPCs in peritumor tissue at the 16th week in a HCC model. Furthermore, it is confirmed that the macrophage-derived TWEAK/Fn14 promoted the expression of inhibitor of differentiation-1 (ID1) in HPCs via NF-κB signaling and a high level of ID1 induced aberrant differentiation of HPCs. Mechanistically, ID1 suppressed differentiation and promoted proliferation in HPCs through the inhibition of HNF4α and Rap1GAP transcriptions. Finally, scRNA sequencing of HCC patients and investigation of clinical specimens also verified that the expression of ID1 is correlated with aberrant differentiation of HPCs into cancer stem cells, patients with high levels of ID1 in HPCs showed a poorer prognosis. This study provides important intervention targets and a theoretical basis for the clinical diagnosis and treatment of HCC.

Therapeutic targeting of prenatal pontine ID1 signaling in diffuse midline glioma.

BACKGROUND: Diffuse midline gliomas (DMG) are highly invasive brain tumors with rare survival beyond two years past diagnosis and limited understanding of the mechanism behind tumor invasion. Previous reports demonstrate upregulation of the protein ID1 with H3K27M and ACVR1 mutations in DMG, but this has not been confirmed in human tumors or therapeutically targeted. METHODS: Whole exome, RNA, and ChIP-sequencing was performed on the ID1 locus in DMG tissue. Scratch-assay migration and transwell invasion assays of cultured cells were performed following shRNA-mediated ID1-knockdown. In vitro and in vivo genetic and pharmacologic [cannabidiol (CBD)] inhibition of ID1 on DMG tumor growth was assessed. Patient-reported CBD dosing information was collected. RESULTS: Increased ID1 expression in human DMG and in utero electroporation (IUE) murine tumors is associated with H3K27M mutation and brainstem location. ChIP-sequencing indicates ID1 regulatory regions are epigenetically active in human H3K27M-DMG tumors and prenatal pontine cells. Higher ID1-expressing astrocyte-like DMG cells share a transcriptional program with oligo/astrocyte-precursor cells (OAPCs) from the developing human brain and demonstrate upregulation of the migration regulatory protein SPARCL1. Genetic and pharmacologic (CBD) suppression of ID1 decreases tumor cell invasion/migration and tumor growth in H3.3/H3.1K27M PPK-IUE and human DIPGXIIIP* in vivo models of pHGG. The effect of CBD on cell proliferation appears to be non-ID1 mediated. Finally, we collected patient-reported CBD treatment data, finding that a clinical trial to standardize dosing may be beneficial. CONCLUSIONS: H3K27M-mediated re-activation of ID1 in DMG results in a SPARCL1+ migratory transcriptional program that is therapeutically targetable with CBD.

ID1 and CEBPA coordinate epidermal progenitor cell differentiation.

The regulatory circuits that coordinate epidermal differentiation during development are still not fully understood. Here, we report that the transcriptional regulator ID1 is enriched in mouse basal epidermal progenitor cells and find ID1 expression to be diminished upon differentiation. In utero silencing of Id1 impairs progenitor cell proliferation, leads to precocious delamination of targeted progenitor cells and enables differentiated keratinocytes to retain progenitor markers and characteristics. Transcriptional profiling suggests that ID1 acts by mediating adhesion to the basement membrane while inhibiting spinous layer differentiation. Co-immunoprecipitation reveals ID1 binding to transcriptional regulators of the class I bHLH family. We localize bHLH Tcf3, Tcf4 and Tcf12 to epidermal progenitor cells during epidermal stratification and establish TCF3 as a downstream effector of ID1-mediated epidermal proliferation. Finally, we identify crosstalk between CEBPA, a known mediator of epidermal differentiation, and Id1, and demonstrate that CEBPA antagonizes BMP-induced activation of Id1. Our work establishes ID1 as a key coordinator of epidermal development, acting to balance progenitor proliferation with differentiation and unveils how functional crosstalk between CEBPA and Id1 orchestrates epidermal lineage progression.

RSL3 triggers glioma stem cell differentiation via the Tgm2/AKT/ID1 signaling axis.

RSL3 is a synthetic molecule that inactivates glutathione peroxidase 4 to induce ferroptosis. However, its effect on glioma stem cells (GSC) remains unclear. In this study, we found that RSL3 significantly suppressed GSC proliferation and induced their differentiation into astrocytes, which was accompanied by the downregulation of stemness-related markers, including Nestin and Sox2. Combined transcriptome and proteome analyses further revealed that RSL3 promoted GSC differentiation by suppressing transglutaminase 2 (Tgm2), but not by ferroptosis-related pathways. Tgm2 overexpression in CSC2078 cells rescued the changes in stemness-related markers and differentiation caused by RSL3, which was mediated by inhibitor of DNA binding 1 (ID1) activation. Further studies identified ID1 as a downstream signaling target of Tgm2. Blocking the phosphoinositide-3 kinase (PI3K)/Akt pathway with LY294002 suppressed PI3K, p-Akt, and ID1 levels but not Tgm2. Tgm2 overexpression abrogated the changes in PI3K, p-Akt, and ID1 levels caused by LY294002. Taken together, we demonstrate that RSL3 does not induce ferroptosis; instead, it inhibits GSC proliferation and triggers their differentiation by suppressing the Tgm2/Akt/ID1 signaling axis.

ID1 marks the tumorigenesis of pancreatic ductal adenocarcinoma in mouse and human.

Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly disease that has an increasing death rate but no effective treatment to now. Although biological and immunological hallmarks of PDAC have been frequently reported recently, early detection and the particularly aggressive biological features are the major challenges remaining unclear. In the current study, we retrieved multiple scRNA-seq datasets and illustrated the genetic programs of PDAC development in genetically modified mouse models. Notably, the transcription levels of Id1 were elevated specifically along with the PDAC development. Pseudotime trajectory analysis revealed that Id1 was closely correlated with the malignancy of PDAC. The gene expression patterns of human PDAC cells were determined by the comparative analysis of the scRNA-seq data on human PDAC and normal pancreas tissues. ID1 levels in human PDAC cancer cells were dramatically increased compared to normal epithelial cells. ID1 deficiency in vitro significantly blunt the invasive tumor-formation related phenotypes. IPA analysis on the differentially expressed genes suggested that EIF2 signaling was the core pathway regulating the development of PDAC. Blocking EFI2 signaling remarkably decreased the expression of ID1 and attenuated the tumor-formation related phenotypes. These observations confirmed that ID1 was regulated by EIF2 signaling and was the critical determinator of PDAC development and progression. This study suggests that ID1 is a potential malignant biomarker of PDAC in both mouse models and human and detecting and targeting ID1 may be a promising strategy to treat or even rescue PDAC.

5-Demethylnobiletin Inhibits Cell Proliferation, Downregulates ID1 Expression, Modulates the NF-κB/TNF-α Pathway and Exerts Antileukemic Effects in AML Cells.

Acute myeloid leukemia (AML) is characterized by the dysregulation of hematopoietic cell proliferation, resulting in the accumulation of immature myeloid cells in bone marrow. 5-Demethylnobiletin (5-demethyl NOB), a citrus 5-hydroxylated polymethoxyflavone, has been reported to exhibit various bioactivities, such as antioxidant, anti-inflammatory and anticancer properties. In this study, we investigated the antileukemic effects of 5-demethyl NOB and its underlying molecular mechanisms in human AML cells. We found that 5-demethyl NOB (20−80 µM) significantly reduced human leukemia cell viability, and the following trend of effectiveness was observed: THP-1 ≈ U-937 > HEL > HL-60 > K562 cells. 5-Demethyl NOB (20 and 40 µM) modulated the cell cycle through the regulation of p21, cyclin E1 and cyclin A1 expression and induced S phase arrest. 5-Demethyl NOB also promoted leukemia cell apoptosis and differentiation. Microarray-based transcriptome, Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) of differentially expressed genes (DEGs) analysis showed that the expression of inhibitor of differentiation/DNA binding 1 (ID1), a gene associated with the GO biological process (BP) cell population proliferation (GO: 0008283), was most strongly suppressed by 5-demethyl NOB (40 µM) in THP-1 cells. We further demonstrated that 5-demethyl NOB-induced ID1 reduction was associated with the inhibition of leukemia cell growth. Moreover, DEGs involved in the hallmark gene set NF-κB/TNF-α signaling pathway were markedly enriched and downregulated by 5-demethyl NOB. Finally, we demonstrated that 5-demethyl NOB (20 and 40 µM), combined with cytarabine, synergistically reduced THP-1 and U-937 cell viability. Our current findings support that 5-demethyl NOB dramatically suppresses leukemia cell proliferation and may serve as a potential phytochemical for human AML chemotherapy.

ID proteins promote the survival and primed-to-naive transition of human embryonic stem cells through TCF3-mediated transcription.

Inhibition of DNA binding proteins 1 and 3 (ID1 and ID3) are important downstream targets of BMP signalling that are necessary for embryonic development. However, their specific roles in regulating the pluripotency of human embryonic stem cells (hESCs) remain unclear. Here, we examined the roles of ID1 and ID3 in primed and naive-like hESCs and showed that ID1 and ID3 knockout lines (IDs KO) exhibited decreased survival in both primed and naive-like state. IDs KO lines in the primed state also tended to undergo pluripotent dissolution and ectodermal differentiation. IDs KO impeded the primed-to-naive transition (PNT) of hESCs, and overexpression of ID1 in primed hESCs promoted PNT. Furthermore, single-cell RNA sequencing demonstrated that ID1 and ID3 regulated the survival and pluripotency of hESCs through the AKT signalling pathway. Finally, we showed that TCF3 mediated transcriptional inhibition of MCL1 promotes AKT phosphorylation, which was confirmed by TCF3 knockdown in KO lines. Our study suggests that IDs/TCF3 acts through AKT signalling to promote survival and maintain pluripotency of both primed and naive-like hESCs.

Long non-coding RNA nuclear enriched abundant transcript 1 (NEAT1) modulates inhibitor of DNA binding 1 (ID1) to facilitate papillary thyroid carcinoma development by sponging microRNA-524-5p.

Long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) exerts a pro-oncogenic role in several cancers, whereas its underlying regulatory mechanism in papillary thyroid carcinoma (PTC) progression remains unknown. This research mainly explored the roles of NEAT1 in PTC development. Quantitative real-time polymerase-chain reaction (qRT-PCR) was applied to measure NEAT1, miR-524-5p, and inhibitor of DNA binding 1 (ID1) expression in PTC tissues and cells. Western blot was conducted for detecting the protein levels. MTT, transwell, and flow cytometry assays were applied to assess cell proliferation, metastasis, and apoptosis in PTC cells in vitro. The PTC xenograft tumor model was used for investigating the role of NEAT1 in vivo. Dual-luciferase reporter assay was utilized for confirming the interaction between miR-524-5p and NEAT1 or ID1. In PTC tissues and cells, NEAT1 was significantly up-regulated. NEAT1 silencing blocked cell proliferation, metastasis, and facilitated apoptosis in vitro and impeded xenograft tumor growth in vivo. Bioinformatics prediction revealed the existence of binding sites between NEAT1 and miR-524-5p. Besides, ID1 was confirmed as a direct target to miR-524-5p, and the enhancement of ID1 reversed the regulation of miR-524-5p upregulation on cell progression. In addition, NEAT1 promoted PTC development by regulating ID1 expression via sponging miR-524-5p in PTC. In summary, we demonstrate that NEAT1 advanced the process of PTC by miR-524-5p/ID1 axis, which may enhance our comprehension of PTC pathogenesis.

Deubiquitinase USP8 increases ID1 stability and promotes esophageal squamous cell carcinoma tumorigenesis.

ID1 is a labile protein implicated in the development and progression of several malignant tumors, but the mechanisms regulating ID1 stability and function in human esophageal squamous cell carcinoma (ESCC) are largely unclear. In this study, we performed an immunoprecipitation assay to screen for deubiquitinases that may interact with ID1 and identified USP8 as a novel deubiquitinase for ID1. USP8 interacts with ID1, and increases the protein level and stability of ID1 by reducing its ubiquitination. Ectopic expression of USP8 promotes ESCC tumorigenesis by suppressing ID1 degradation, whereas knockdown of USP8 results in the opposite phenotypes in vitro and in vivo. Moreover, we found that TXNIP is a novel downstream target of ID1, and USP8 promotes ESCC tumorigenesis by activating the ID1-TXNIP pathway. Increased expression of USP8 and ID1 positively correlates with reduced TXNIP expression in ESCC tissues and predicts an advanced tumor stage. Overall, our data indicate that USP8 is a novel deubiquitinase for ID1 and show the importance of the USP8-ID1-TXNIP axis in promoting ESCC tumorigenesis.

BMP9-ID1 Signaling Activates HIF-1α and VEGFA Expression to Promote Tumor Angiogenesis in Hepatocellular Carcinoma.

Since hepatocellular carcinoma (HCC) is a typical hypervascular malignant tumor with poor prognosis, targeting angiogenesis is an important therapeutic strategy for advanced HCC. Involvement of bone morphologic protein 9 (BMP9), a transforming growth factor-beta superfamily member, has recently been reported in the development of liver diseases and angiogenesis. Here, we aimed to elucidate the role of BMP9 signaling in promoting HCC angiogenesis and to assess the antiangiogenic effect of BMP receptor inhibitors in HCC. By analyzing HCC tissue gene expression profiles, we found that BMP9 expression was significantly correlated with angiogenesis-associated genes, including HIF-1α and VEGFR2. In vitro, BMP9 induced HCC cell HIF-1α/VEGFA expression and VEGFA secretion. Silencing of the inhibitor of DNA-binding protein 1 (ID1), a transcription factor targeted by BMP9 signaling, suppressed BMP9-induced HIF-1α/VEGFA expression and VEGFA secretion, resulting in decreased human umbilical vein endothelial cell (HUVEC) lumen formation. BMP receptor inhibitors, which inhibit BMP9-ID1 signaling, suppressed BMP9-induced HIF-1α/VEGFA expression, VEGFA secretion, and HUVEC lumen formation. In vivo, the BMP receptor inhibitor LDN-212854 successfully inhibited HCC tumor growth and angiogenesis by inhibiting BMP9-ID1 signaling. In summary, BMP9-ID1 signaling promotes HCC angiogenesis by activating HIF-1α/VEGFA expression. Thus, targeting BMP9-ID1 signaling could be a pivotal therapeutic option for advanced HCC.

Reconstituted HDL-apoE3 promotes endothelial cell migration through ID1 and its downstream kinases ERK1/2, AKT and p38 MAPK.

INTRODUCTION: Atherosclerotic Coronary Artery Disease (ASCAD) is the leading cause of mortality worldwide. Novel therapeutic approaches aiming to improve the atheroprotective functions of High Density Lipoprotein (HDL) include the use of reconstituted HDL forms containing human apolipoprotein A-I (rHDL-apoA-I). Given the strong atheroprotective properties of apolipoprotein E3 (apoE3), rHDL-apoE3 may represent an attractive yet largely unexplored therapeutic agent. OBJECTIVE: To evaluate the atheroprotective potential of rHDL-apoE3 starting with the unbiased assessment of global transcriptome effects and focusing on endothelial cell (EC) migration as a critical process in re-endothelialization and atherosclerosis prevention. The cellular, molecular and functional effects of rHDL-apoE3 on EC migration-associated pathways were assessed, as well as the potential translatability of these findings in vivo. METHODS: Human Aortic ECs (HAEC) were treated with rHDL-apoE3 and total RNA was analyzed by whole genome microarrays. Expression and phosphorylation changes of key EC migration-associated molecules were validated by qRT-PCR and Western blot analysis in primary HAEC, Human Coronary Artery ECs (HCAEC) and the human EA.hy926 EC line. The capacity of rHDL-apoE3 to stimulate EC migration was assessed by wound healing and transwell migration assays. The contribution of MEK1/2, PI3K and the transcription factor ID1 in rHDL-apoE3-induced EC migration and activation of EC migration-related effectors was assessed using specific inhibitors (PD98059: MEK1/2, LY294002: PI3K) and siRNA-mediated gene silencing, respectively. The capacity of rHDL-apoE3 to improve vascular permeability and hypercholesterolemia in vivo was tested in a mouse model of hypercholesterolemia (apoE KO mice) using Evans Blue assays and lipid/lipoprotein analysis in the serum, respectively. RESULTS: rHDL-apoE3 induced significant expression changes in 198 genes of HAEC mainly involved in re-endothelialization and atherosclerosis-associated functions. The most pronounced effect was observed for EC migration, with 42/198 genes being involved in the following EC migration-related pathways: 1) MEK/ERK, 2) PI3K/AKT/eNOS-MMP2/9, 3) RHO-GTPases, 4) integrin. rHDL-apoE3 induced changes in 24 representative transcripts of these pathways in HAEC, increasing the expression of their key proteins PIK3CG, EFNB2, ID1 and FLT1 in HCAEC and EA.hy926 cells. In addition, rHDL-apoE3 stimulated migration of HCAEC and EA.hy926 cells, and the migration was markedly attenuated in the presence of PD98059 or LY294002. rHDL-apoE3 also increased the phosphorylation of ERK1/2, AKT, eNOS and p38 MAPK in these cells, while PD98059 and LY294002 inhibited rHDL-apoE3-induced phosphorylation of ERK1/2, AKT and p38 MAPK, respectively. LY had no effect on rHDL-apoE3-mediated eNOS phosphorylation. ID1 siRNA markedly decreased EA.hy926 cell migration by inhibiting rHDL-apoE3-triggered ERK1/2 and AKT phosphorylation. Finally, administration of a single dose of rHDL-apoE3 in apoE KO mice markedly improved vascular permeability as demonstrated by the reduced concentration of Evans Blue dye in tissues such as the stomach, the tongue and the urinary bladder and ameliorated hypercholesterolemia. CONCLUSIONS: rHDL-apoE3 significantly enhanced EC migration in vitro, predominantly via overexpression of ID1 and subsequent activation of MEK1/2 and PI3K, and their downstream targets ERK1/2, AKT and p38 MAPK, respectively, and improved vascular permeability in vivo. These novel insights into the rHDL-apoE3 functions suggest a potential clinical use to promote re-endothelialization and retard development of atherosclerosis.

Screening of host genes regulated by ID1 and ID3 proteins during foot-and-mouth disease virus infection.

Foot-and-mouth disease virus (FMDV) is an important pathogen that harms cloven-hoofed animals and has caused serious losses to livestock production since its discovery. Furthermore, inhibitor of DNA binding (ID) proteins have been thoroughly studied in tumorigenesis, differentiation and metastasis, but its role in viral infection is rarely known. In this study, three gene knockout cell lines ID1 KO, ID3 KO, ID1/3 KO were obtained based on BHK-21 cells. We found that ID1 and ID3 genes single or double knockout promote the replication of FMDV. Moreover, compared with negative control cells during virus infection, there were 551 up-regulated genes and 1222 down-regulated genes in the ID1 KO cell line; 916 up-regulated genes and 1845 down-regulated genes in the ID3 KO cell line; 810 up-regulated genes and 1566 down-regulated genes in ID1/3 KO cell line. Further genes expression patterns verification results also showed a good correlation between the data of RT-qRCR and RNA-seq. These findings provide a basis for studying the relevant mechanisms between host genes and ID genes during FMDV infection.

Transcriptomic characteristics and impaired immune function of patients who retest positive for SARS-CoV-2 RNA.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has become a global public health crisis. Some patients who have recovered from COVID-19 subsequently test positive again for SARS-CoV-2 RNA after discharge from hospital. How such retest-positive (RTP) patients become infected again is not known. In this study, 30 RTP patients, 20 convalescent patients, and 20 healthy controls were enrolled for the analysis of immunological characteristics of their peripheral blood mononuclear cells. We found that absolute numbers of CD4+ T cells, CD8+ T cells, and natural killer cells were not substantially decreased in RTP patients, but the expression of activation markers on these cells was significantly reduced. The percentage of granzyme B-producing T cells was also lower in RTP patients than in convalescent patients. Through transcriptome sequencing, we demonstrated that high expression of inhibitor of differentiation 1 (ID1) and low expression of interferon-induced transmembrane protein 10 (IFITM10) were associated with insufficient activation of immune cells and the occurrence of RTP. These findings provide insight into the impaired immune function associated with COVID-19 and the pathogenesis of RTP, which may contribute to a better understanding of the mechanisms underlying RTP.