Id1 Levels Research Articles

Integrated analysis of microRNA and mRNA expression profiles in homozygous familial hypercholesterolemia patients and validation of atherosclerosis associated critical regulatory network

Homozygous familial hypercholesterolemia (HoFH) is a rare, life-threatening genetic disorder characterized by an extremely elevated serum level of low-density lipoprotein cholesterol (LDL-C) and accelerated premature atherosclerotic cardiovascular diseases (ASCVD). However, the detailed mechanism of how the pathogenic mutations of HoFH trigger the acceleration of ASCVD is not well understood. Therefore, we performed high-throughput RNA and small RNA sequencing on the peripheral blood RNA samples of six HoFH patients and three healthy controls. The gene and miRNA expression differences were analyzed, and seven miRNAs and six corresponding genes were screened out through regulatory network analysis. Validation through quantitative PCR of genes and miRNAs from 52 HoFH patients and 20 healthy controls revealed that the expression levels of hsa-miR-486-3p, hsa-miR-941, and BIRC5 were significantly upregulated in HoFH, while ID1, PLA2G4C, and CACNA2D2 were downregulated. Spearman correlation analysis found that the levels of ID1, hsa-miR-941, and hsa-miR-486-3p were significantly correlated with additional ASCVD risk factors in HoFH patients. This study represents the first integrated analysis of transcriptome and miRNA expression profiles in patients with HoFH, a rare disease, and as a result, six differentially expressed miRNAs/genes that may be related to atherosclerosis in HoFH are reported. The miRNA–mRNA regulatory network may be the critical regulation mechanism by which ASCVD is accelerated in HoFH.

Harmine targets inhibitor of DNA binding-2 and activator protein-1 to promote preosteoclast PDGF-BB production.

Osteoporosis is one of the most common metabolic bone diseases affecting millions of people. We previously found that harmine prevents bone loss in ovariectomized mice via increasing preosteoclast platelet‐derived growth factor‐BB (PDGF‐BB) production and type H vessel formation. However, the molecular mechanisms by which harmine promotes preosteoclast PDGF‐BB generation are still unclear. In this study, we revealed that inhibitor of DNA binding‐2 (Id2) and activator protein‐1 (AP‐1) were important factors implicated in harmine‐enhanced preosteoclast PDGF‐BB production. Exposure of RANKL‐induced Primary bone marrow macrophages (BMMs), isolated from tibiae and femora of mice, to harmine increased the protein levels of Id2 and AP‐1. Knockdown of Id2 by Id2‐siRNA reduced the number of preosteoclasts as well as secretion of PDGF‐BB in RANKL‐stimulated BMMs administrated with harmine. Inhibition of c‐Fos or c‐Jun (components of AP‐1) both reversed the stimulatory effect of harmine on preosteoclast PDGF‐BB production. Dual‐luciferase reporter assay analyses determined that PDGF‐BB was the direct target of AP‐1 which was up‐regulated by harmine treatment. In conclusion, our data demonstrated a novel mechanism involving in the production of PDGF‐BB increased by harmine, which may provide potential therapeutic targets for bone loss diseases.

Roles of NR5A1 and NR5A2 in the regulation of steroidogenesis by Clock gene and bone morphogenetic proteins by human granulosa cells.

The functional role of the transcription factors NR5A1 and NR5A2 and their interaction with Clock gene and bone morphogenetic proteins (BMPs) were investigated in human granulosa KGN cells. Treatment with BMP-15 and GDF-9 suppressed forskolin (FSK)-induced steroidogenesis as shown by the mRNA expression levels of StAR and P450scc but not the mRNA expression level of P450arom. Of interest, treatment with BMP-15 and GDF-9 also suppressed FSK-induced NR5A2 mRNA expression. Treatment with BMP-15 suppressed NR5A2 mRNA and protein expression but increased Clock mRNA and protein expression levels by granulosa cells. The mRNA expression levels of NR5A1, but not those of NR5A2, were positively correlated with the levels of Clock mRNA, while the mRNA levels of Id-1, the target gene of BMP signaling, were positively correlated with those of NR5A1 but not with those of NR5A2. It was also demonstrated that the mRNA expression levels of NR5A1 were positively correlated with those of P450arom and 3βHSD, whereas the mRNA expression level of NR5A2 was correlated with those of StAR and P450scc. Furthermore, inhibition of Clock gene expression by siRNA attenuated the expression of NR5A1, and the mRNA levels of Clock gene were significantly correlated with those of NR5A1. Collectively, the results suggested a novel mechanism by which Clock gene expression induced by BMP-15 is functionally linked to the expression of NR5A1, whereas NR5A2 expression is suppressed by BMP-15 in granulosa cells. The interaction between Clock NR5A1/NR5A2 and BMP-15 is likely to be involved in the fine-tuning of steroidogenesis by ovarian granulosa cells.

Prognostic effects of the expression of inhibitor of DNA-binding family members on patients with lung adenocarcinoma.

The basic helix-loop-helix (bHLH) transcription factors are negatively regulated by inhibitor of DNA-binding (ID) proteins. Several studies have demonstrated that ID family proteins are dysregulated in a variety of cancer types, including in lung adenocarcinoma (LUAD). In current study, the prognostic value of ID family members was evaluated by investigating publicly accessible databases, including Oncomine, Kaplan-Meier plotter, UALCAN and the Human Protein Atlas. It was observed that the mRNA expression of all ID members was downregulated in LUAD tumor tissues compared with those in normal tissues according to the Oncomine and UALCAN databases. Additionally, increased mRNA expression levels of ID2 and ID1 were associated with improved and poorer survival time, respectively. Notably, ID3 and ID4 expression was not associated with survival in patients with LUAD. At the protein level, high ID2 significantly predicted an improved survival outcome while high ID1 is associated with shorter survival time. Thus, the results indicate that the ID proteins, particularly ID2, exhibit significant prognostic value in LUAD. More studies are required to elucidate the underlying molecular mechanisms behind the role of the ID family in the development of LUAD.

Therapeutic Efficacy of Immune Stimulatory Thymidine Kinase and fms-like Tyrosine Kinase 3 Ligand (TK/Flt3L) Gene Therapy in a Mouse Model of High-Grade Brainstem Glioma.

Diffuse intrinsic pontine glioma (DIPG) bears a dismal prognosis. A genetically engineered brainstem glioma model harboring the recurrent DIPG mutation, Activin A receptor type I (ACVR1)-G328V (mACVR1), was developed for testing an immune-stimulatory gene therapy. We utilized the Sleeping Beauty transposase system to generate an endogenous mouse model of mACVR1 brainstem glioma. Histology was used to characterize and validate the model. We performed RNA-sequencing analysis on neurospheres harboring mACVR1. mACVR1 neurospheres were implanted into the pons of immune-competent mice to test the therapeutic efficacy and toxicity of immune-stimulatory gene therapy using adenoviruses expressing thymidine kinase (TK) and fms-like tyrosine kinase 3 ligand (Flt3L). mACVR1 neurospheres expressing the surrogate tumor antigen ovalbumin were generated to investigate whether TK/Flt3L treatment induces the recruitment of tumor antigen-specific T cells. Histologic analysis of mACVR1 tumors indicates that they are localized in the brainstem and have increased downstream signaling of bone morphogenetic pathway as demonstrated by increased phospho-smad1/5 and Id1 levels. Transcriptome analysis of mACVR1 neurosphere identified an increase in the TGFβ signaling pathway and the regulation of cell differentiation. Adenoviral delivery of TK/Flt3L in mice bearing brainstem gliomas resulted in antitumor immunity, recruitment of antitumor-specific T cells, and increased median survival (MS). This study provides insights into the phenotype and function of the tumor immune microenvironment in a mouse model of brainstem glioma harboring mACVR1. Immune-stimulatory gene therapy targeting the hosts' antitumor immune response inhibits tumor progression and increases MS of mice bearing mACVR1 tumors.

Comprehensive analysis of inhibitor of differentiation/DNA-binding gene family in lung cancer using bioinformatics methods

The inhibitor of differentiation/DNA-binding (ID) is a member of the helix–loop–helix (HLH) transcription factor family, and plays a role in tumorigenesis, invasiveness and angiogenesis. The aims were to investigate the expression patterns and prognostic values of individual ID family members in lung cancer, and the potential functional roles. The expression levels of ID family were assessed using the Oncomine online database and GEPIA database. Furthermore, the prognostic value of ID family members was evaluated using the Kaplan–Meier plotter database. The genetic mutations of ID family members were investigated using the cBioPortal database. Moreover, enrichment analysis was performed using STRING database and Funrich software. It was found that all the ID family members were significantly down-regulated in lung cancer. Prognostic results indicated that low mRNA expression levels of ID1 or increased mRNA expression levels of ID2/3/4 were associated with improved overall survival, first progression and post progression survival. Additionally, genetic mutations of ID family members were identified in lung cancer, and it was suggested that amplification and deep deletion were the main mutation types. Furthermore, functional enrichment analysis results suggested that ID1/2/4 were significantly enriched in ‘regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism’ for biological process, ‘transcription factor activity’ for molecular function and ‘HLH domain’ for protein domain. However, it was found that ID3 was not enriched in the above functions. The aberrant expression of ID family members may affect the occurrence and prognosis of lung cancer, and may be related to cell metabolism and transcriptional regulation.

Id-1 Promotes Reendothelialization In The Early Phase After Vascular Injury Through Activation Of NFkB/survivin Signaling Pathway.

BackgroundPercutaneous coronary intervention (PCI) treatment can benefit patients, but also cause irreversible mechanical damage to the vascular endothelium, ultimately leading to restenosis of the target vessel. Thus, achieving rapid re-endothelialization and restoring the integrity of the vascular endothelium and function plays an important role in inhibiting neointimal hyperplasia and preventing restenosis. Id1 (inhibitor of DNA binding/differentiation factor 1) plays an important role in promoting cell proliferation and angiogenesis.Study objectiveThis study aims to investigate the relationship between Id1 and NFκB/survivin signaling pathways and their role in injured vascular repair by establishing a rat carotid balloon injury model.MethodsThe carotid artery model of rat balloon injury was established. The injured common carotid artery was obtained at different time points after vascular injury. RNA and protein were extracted and the mRNA and protein expression levels of Id1, NFκB and survivin were detected in vascular injury. The NFκB blocker BAY 11–7082 and survivin blocker YM155 were used and the effects of Id1, NFκB, survivin mRNA and protein expression, revascularization of blood vessels and neointimal responsiveness after vascular injury were observed in the vascular tissues of Ad-Id1 transfected balloon injury.ResultsId1, NFκB and survivin were expressed in injured rat carotid arteries. Overexpression of Id1 promoted re-endothelialization of injured vessels through NFκB/survivin signaling pathway, inhibited early vascular endometrial reactive hyperplasia; blocked NFκB the/survivin signaling pathway attenuates the re-endothelialization of Ad-Id1 and the early endothelium of Ad-Id1. Blocking the NFκB/survivin signaling pathway attenuates the re-endothelialization and early reactive hyperplasia of vascular intima of Ad-Id1.ConclusionNF-kappa B/survivin signaling pathway may play an important role in Id1 promoting vascular re-endothelialization, inhibiting neointimal hyperplasia and preventing vascular restenosis.

MA17.11 High Sensitivity to PD-1 Blockade Therapy After Ld1 Depletion in KRAS-Driven Lung Cancer Through CD8+/CD3+ Tumor Infiltration and PD-L1 Induction

PD-1/PDL-1 inhibitors are approved for non-small cell lung cancer (NSCLC). However, many patients do not benefit and therapeutic combinations are under investigation. We have previously described Id1, involved in proliferation, angiogenesis and immunosuppression, as a prognostic factor in lung adenocarcinoma (LUAD) (Ponz-Sarvise, Clin Cancer Res 2011), Id1’s role in lung cancer metastasis (Castanon, Cancer Letters 2017) and more recently shown that Id1 sustains mutant KRAS-driven progression and metastasis in NSCLC (Roman, Cancer Res 2019). In a previous syngeneic murine lung cancer model with depleted levels of Id1 using Id1-/- and Id1 wildtype C57BL/6 mice inoculated with Lewis Lung Carcinoma (LLC), we tested a combined therapeutic strategy targeting PD-1 and Id1, showing impaired tumor growth and increased survival (Gil-Bazo, presented at WCLC 2018). Here we study a combined strategy targeting PD-1 and Id1 in a KRAS-mutant murine LUAD model and the immune-related mechanisms involved. First, a correlation between Id1 and PD-L1 mRNA expression was studied in mutant and wild-type KRAS LUAD cohorts from The Cancer Genome Atlas data set (TCGA). Secondly, a syngeneic tumor model using Balb/c mice through subcutaneous injection of KRAS-mutant LUAD (Lacun3) cells and Id1-silenced Lacun3 (Id1sh) cells. In vitro, proliferation was measured in both cell lines through MTS assays. IFNg-induced PD-L1 expression in both cell lines and flow cytometry was used to evaluate its mechanistic effects on the immune response. After tumor cells injection, mice were treated with an anti-PD-1 (RMP-1-14) monoclonal antibody or PBS, i.p. Tumor volumes according to Id1 status in tumor cells and the treatment administered were quantified. Vectra 3.0™ multispectral microscopy was used to characterize the tumor associated immune cells in paraffin-embedded tissues from our previous syngeneic murine lung cancer model using Id1-/- and Id1 wildtype C57BL/6 mice inoculated with LLC in which the combined blockade had been reported as effective. Immune marker antibodies were used to study expression of CD3, CD4 and CD8. An inverse, moderate and statistically significant correlation between Id1 and PD-L1 expression in mutant and wild-type KRAS LUAD cohorts from TCGA was found in both cohorts (-0.367 and -0.351, respectively, p<0.001), indicating that Id1 depletion may lead to PD-L1 expression induction. In vitro assays showed that Id1 silencing reduced Lacun3 cells proliferation (p<0.001). Up-regulation of surface PD-L1 expression occurred in Id1sh cells, but not in Lacun3 cells, after receiving IFNg (p=0.0022). Mechanistically, in the syngeneic murine model, Id1 inhibition in the injected cells, combined with anti-PD-1 treatment, significantly induced a tumor growth impairment (p<0.001). An intense CD8+ and CD3+ immune cell infiltration was observed in LLC Id1-/- C57BL/6 mice treated with anti-PD1 (p<0.05 for CD3+ TILS), compared the control groups, possibly explaining the dramatic tumor growth impairment previously shown on the treated animals. Id1 silencing may induce PD-L1 overexpression according to in silico and in vitro results. Id1 and PD-1 combined blockade in our KRAS-mutant syngeneic murine LUAD model significantly impaired tumor growth, compared to each strategy alone. A significantly increased CD3+ and CD8+ tumor infiltration and IFNg-induced PD-L1 tumor expression after the combined blockade may explain these findings.

Abstract 4306: JunD-induced cell proliferation requires MYC signaling in prostate cancer cells

Abstract JunD, a member of the AP-1 transcription factor family, is involved in a variety of biological processes including cell proliferation, inflammation, differentiation, apoptosis, and carcinogenesis. We previously showed that JunD is essential for cell proliferation and demonstrated that the knock-down (KD) of JunD in PCa cells resulted in cell cycle arrest in G1-phase concomitant with a decrease in the levels of Id1, c-MYC, and Ki67, but an increased in p21 protein levels. Furthermore, the over-expression of JunD significantly increased proliferation in these cells suggesting that JunD regulates the expression of genes which are required for cell cycle progression. In this present study, employing gene expression profiling, quantitative proteomics, and validation approaches, we demonstrated that JunD KD is associated with distinct gene and protein expression patterns. Comparison integrative analysis by Ingenuity Pathway Analysis (IPA) identified 1) cell cycle control/regulation as the top canonical pathway whose members exhibited a significant decrease in their expression following JunD KD including PRDX3, PEA15, KIF2C, and CDK2, and 2) JunD target genes are involved in cell proliferation, with MYC as the key downstream regulator. Conversely, JunD over-expression induced cell proliferation and the expression of the above JunD target genes including c-MYC. When treated with JQ1, a c-MYC inhibitor, these cells exhibited a significant reduction in cell proliferation and a decrease in JunD target genes. Our data suggest that JunD-mediated cell proliferation involves MYC signaling and inhibiting its target genes may be an effective approach to blocking prostate carcinogenesis Citation Format: Bethtrice Thompson (Elliott), Ana Cecilia Millena, Lilya Matyunina, Mengnan Zhang, Jin Zou, Guangdi Wang, Qiang Zhang, Nathan Bowen, Vanessa Eaton, Gabrielle Webb, Shadyra Thompson, John McDonald, Shafiq Khan. JunD-induced cell proliferation requires MYC signaling in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4306.

CCN2\u2013MAPK\u2013Id-1 loop feedback amplification is involved in maintaining stemness in oxaliplatin-resistant hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Chemotherapy is an alternative treatment for advanced HCCs, but chemo-resistance prevents cancer therapies from achieving stable and complete responses. Understanding the underlying mechanisms in chemo-resistance is critical to improve the efficacy of HCC.MethodsThe expression levels of Id-1 and CCN2 were detected in large cohorts of HCCs, and functional analyses of Id-1 and CCN2 were performed both in vitro and in vivo. cDNA microarrays were performed to evaluate the alterations of expression profiling of HCC cells with overexpression of CCN2. Finally, the role of downstream signaling of MAPK/Id-1 signaling pathway in oxaliplatin resistance were also explored.ResultsThe increased expression of Id-1 and CCN2 were closely related to oxaliplatin resistance in HCC. Upregulation of CCN2 and Id-1 was independently associated with shorter survival and increased recurrence in HCC patients, and significantly enhanced oxaliplatin resistance and promoted lung metastasis in vivo, whereas knock-down of their expression significantly reversed the chemo-resistance and inhibited HCC cell stemness. cDNA microarrays and PCR revealed that Id-1 and MAPK pathway were the downstream signaling of CCN2. CCN2 significantly enhanced oxaliplatin resistance by activating the MAPK/Id-1 signaling pathway, and Id-1 could upregulate CCN2 in a positive feedback manner.ConclusionsCCN2/MAPK/Id-1 loop feedback amplification is involved in oxaliplatin resistance, and the combination of oxaliplatin with inhibitor of CCN2 or MAPK signaling could provide a promising approach to ameliorating oxaliplatin resistance in HCC.

Inhibitor of DNA Binding 2 (ID2) Plays a Key Tumor Suppressor Role in Promoting Oncogenic Transformation in Multiple Myeloma

Dysregulation of transcriptional control is a common phenomenon associated with oncogenesis. Inhibitors of DNA binding (ID) proteins are critical actors in lymphopoiesis, acting as regulators of transcription through a helix-loop-helix (HLH) domain which enables heterodimerization with basic HLH (bHLH) proteins inhibiting their binding to DNA. ID proteins have been implicated in malignant transformation, but their role in multiple myeloma (MM) is unknown. Here, we evaluated the role of ID proteins in biology and transcriptional dysregulation in MM.We first evaluated the expression of the four ID proteins in normal and malignant plasma cells using RNA sequencing data from a cohort of 360 newly diagnosed MM patients and 16 normal plasma cells. We observed significant downregulation of ID2 in primary patient MM cells in comparison to normal plasma cells (p 0.0013).To study ID2 function in MM cells, we next overexpressed ID2 in 2 MM cell lines (MM1S and NCIH929) and observed a significant decrease in proliferation rate, together with G0/G1 phase cell cycle arrest. We performed RNA-sequencing to evaluate the transcriptomic changes following ID2 overexpression. Gene set enrichment analysis (GSEA) revealed significant downregulation of genes involved in E2F pathway and significant changes in pathways related to immune response, regulation of cell death and cell proliferation. In addition, analysis of upstream cis-regulatory motifs of genes significantly dysregulated in both cell lines (>1.5 fold change) showed a highly significant enrichment for bHLH class I transcription factors (E proteins) binding motifs. Conversely, stable ID2 knockdown in 4 MM cell lines (MM1S, NCIH929, RPMI8226 and KMS11) expressing intermediate levels of ID2, showed an increased proliferation rate, assessed by cell counting, H3-thymidine incorporation and ATP production. RNA-sequencing after ID2 knockdown in MM1S and NCIH929 cells showed 600 common genes upregulated in both cell lines (>1.5 fold change). GSEA revealed upregulation of pathways involved in inflammatory response and epithelial-to-mesenchymal transition, while upstream cis regulatory motifs analysis showed a highly significant enrichment for binding motifs of bHLH class I transcription factors E proteins, in particular Tcf3 (p <0.0001).Next, we sought to investigate the mechanisms involved in ID2 downregulation in MM. Since the role of the microenvironment is critical in myelomagenesis, we evaluated the impact of BM microenvironment on ID2 expression in a co-culture system. Using bone marrow stromal cells (BMSC) derived from MM patients and stromal cell line (HS5) in co-culture with various MM cell lines, we observed that both cell-cell interactions and soluble factors secreted by BMSC or HS5 were able to significantly downregulate ID2 expression at the RNA and protein level. Furthermore, ID2 overexpression in MM cell lines (MM1S and NCIH929) abrogated the impact of BMSC on MM cell proliferation. Next, we evaluated ID2 promoter methylation profile and binding motifs using Sequenom mass array and the assay for transposase-accessible chromatin sequencing (ATAC-seq), respectively. While we didn't observe any increase in methylation of CpG islands located in ID2 promoter in co-culture, explaining ID2 downregulation, we identified several binding motifs corresponding to known driver transcription factors in MM. Especially, we identified SP1 binding motif and we confirmed SP1 binding to ID2 promoter by ChIP-sequencing in MM1S, NCIH929 and U266.These data demonstrate that in MM, ID2 acts as a tumor suppressor by promoting major transcriptomic changes and cell cycle arrest. Bone marrow stromal cells further induce significant downregulation of ID2 in myeloma cells suggesting that ID2/bHLH axis and other ID2 related pathways represent a potential new therapeutic target in myeloma. DisclosuresAnderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; OncoPep: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy. Munshi:OncoPep: Other: Board of director.

ID Proteins May Reduce Aggressiveness of Thyroid Tumors.

ID genes have an important function in the cell cycle, and ID proteins may help identify aggressive tumors, besides being considered promising therapeutic targets. However, their role in thyroid tumors is still poorly understood. We examined ID expression and their correlation with diagnostic and prognostic features aiming to find a clinical application in differentiated thyroid carcinoma (DTC) cases. mRNA levels of ID1, ID2, ID3, and ID4 genes were quantified and their expression was observed by immunohistochemistry in 194 thyroid samples including 68 goiters, 16 follicular adenomas, 75 classic papillary thyroid carcinomas, 18 follicular variants of papillary thyroid carcinoma, 5 follicular thyroid carcinomas, and 1 anaplastic thyroid cancer, besides 11 normal thyroid tissues. DTC patients were managed according to standard protocols and followed up for M = 28 ± 16months. ID2, ID3, and ID4 mRNA levels were higher in benign (2.0 ± 1.9; 0.6 ± 0.6; and 0.7 ± 1.0AU, respectively) than those in malignant nodules (0.30 ± 0.62; 0.3 ± 0.3; and 0.2 ± 0.3AU, respectively, p< 0.0001 for all three genes) and were associated with no extra thyroid invasion or metastasis at diagnosis. ID3 nuclear protein expression was higher in benign than that in malignant cells (5.2 ± 0.9 vs 3.0 ± 1.8AU; p< 0.0001). On the contrary, the cytoplasmic expression of ID3 was higher in malignant than that in benign lesions (5.7 ± 1.5 vs 4.0 ± 1.4AU; p< 0.0001). Our data indicate that ID genes are involved in thyroid tumorigenesis and suggest these genes act impeding the evolution of more aggressive phenotypes. The different patterns of their tissue expression may help identify malignancy and characterize thyroid lesion aggressiveness.

A 3-Protein Expression Signature of Neuroblastoma for Outcome Prediction.

Neuroblastoma (NB) is the most common extracranial solid tumor in children with contrasting outcomes. Precise risk assessment contributes to prognosis prediction, which is critical for treatment strategy decisions. In this study, we developed a 3-protein predictor model, including the neural stem cell marker Msi1, neural differentiation marker ID1, and proliferation marker proliferating cell nuclear antigen (PCNA), to improve clinical risk assessment of patients with NB. Kaplan-Meier analysis in the microarray data (GSE16476) revealed that low expression of ID1 and high expression of Msi1 and PCNA were associated with poor prognosis in NB patients. Combined application of these 3 markers to constitute a signature further stratified NB patients into different risk subgroups can help obtain more accurate prediction performance. Survival prognostic power of age and Msi1_ID1_PCNA signature by receiver operating characteristics analysis showed that this signature predicted more effectively and sensitively compared with classic risk stratification system, compensating for the deficiency of the prediction function of the age. Furthermore, we validated the expressions of these 3 proteins in neuroblastic tumor spectrum tissues by immunohistochemistry revealed that Msi1 and PCNA exhibited increased expression in NB compared with intermedial ganglioneuroblastoma and benign ganglioneuroma, whereas ID1 levels were reduced in NB. In conclusion, we established a robust risk assessment predictor model based on simple immunohistochemistry for therapeutic decisions of NB patients.

Prognostic values of the inhibitor of DNA‑binding family members in breast cancer.

The inhibitor of DNA-binding (ID) proteins are dominant-negative modulators of transcription factors with basic helix-loop-helix (bHLH) structures, which control a variety of genes in cell cycle regulation. An increasing volume of evidence has demonstrated that the deregulated expression of IDs in several types of malignancy, including breast carcinoma, has been proven to serve crucial regulatory functions in tumorigenesis and the development of breast cancer (BC). The present study evaluated the prognostic values of the ID family members by investigating a set of publicly accessible databases, including Oncomine, bc-GenExMiner, Kaplan-Meier plotter and the Human Protein Atlas. The results demonstrated that mRNA levels of distinct IDs exhibited diverse profiles between BC and normal counterparts. The mRNA expression level of ID2 was significantly higher in breast cancer than normal tissues, while the mRNA expression levels of ID1, ID3 and ID4 were significantly lower in breast cancer tissues than in normal tissues. Furthermore, higher mRNA expression levels of ID1 and ID4 were associated with subgroups with lower pathological grades and fewer lymph node metastases. Survival analysis revealed that elevated mRNA levels of ID1 and ID4 predicted an improved survival in all patients with BC. Increased ID1 mRNA levels were associated with higher relapse-free survival rates in all patients with BC, particularly in those with ER positive and Luminal A subtype tumors. Increased ID4 mRNA expression predicted longer survival times in all patients with BC, particularly in those with hormone receptor-positive tumors or those treated with endocrine therapy. These results indicated that IDs are essential prognostic indicators in BC. Future studies on the effect of IDs on the pathogenesis and development of BC are warranted.

Evaluation of changes in the expression of Wnt/β-catenin target genes in mouse reproductive tissues during estrous cycle: An experimental study

The Wingless-type (Wnt)/β-catenin signaling pathway controls cell homeostasis. Reproductive tissues are dynamic in response to steroidal hormone changes. Steroidal hormones are known to control the Wnt/β-catenin pathway, but their role in reproductive tissues remains unknown. The present study aims to investigate the expression patterns of Wnt/β-catenin target genes in mouse reproductive tissues during the normal estrous cycle. In this experimental study, 16 adult NMRI mice were grouped as proestrus, estrus, metestrus, and diestrus according to vaginal smear and histological evaluation of uterine and ovarian tissues. Uterine horns and ovarian tissues were collected. Reverse transcription quantitative polymerase chain reaction was performed to evaluate the expression of Wnt/β-catenin target genes (Myc2, Ppard, Id2, Birc5, and Ascl2) at different stages of the estrous cycle. The expression levels of Id2, Ascl2, and Pprd in uterine tissue were significantly higher at the proestrus phase than at the other stages. Meanwhile, Birc5 expression in uterine tissue was significantly higher at the metestrus stage than at the other stages. Furthermore, Myc2 expression was significantly higher at the diestrus stage than at the estrus and metestrus stages. In the ovarian tissue, the highest expression of Id2, Ascl2, and Birc5 was detected at the proestrus stage, whereas the highest expression of Myc2 and Ppard was observed at the estrus stage. This study showed that Wnt/β-catenin target genes profiles are different among estrous cycle. It seems that different hormonal profiles during estrous cycles play a key role in the expression pattern of Wnt/β-catenin target genes in ovarian and uterine tissue.

Expression and regulation of inhibitor of DNA binding proteins ID1, ID2, ID3, and ID4 at the maternal-conceptus interface in pigs

Inhibitor of DNA binding (ID) proteins, ID1, ID2, ID3, and ID4 are transcriptional regulators that have a helix-loop-helix (HLH) domain but not a basic DNA binding domain. ID proteins inhibit the functions of basic HLH transcription factors and regulate cell proliferation and differentiation. However, the expression and function of ID1, ID2, ID3, and ID4 at the maternal-conceptus interface are not fully understood in pigs. Therefore, we determined the expressions of ID1, ID2, ID3, and ID4 in porcine endometrium, conceptus, and chorioallantoic tissues. ID1, ID2, ID3, and ID4 mRNAs were expressed in the endometrium, with lower levels of ID1, ID2, and ID4 on Day 12 of pregnancy than during the estrous cycle. ID1, ID2, ID3, and ID4 mRNAs were also detected in conceptus and chorioallantoic tissues during pregnancy. ID2 protein was mainly localized to luminal epithelia and weakly to vascular smooth muscle cells in the endometrium and conceptus trophectoderm. Increasing doses of interleukin-1β decreased levels of ID2 mRNA, while estradiol-17β increased levels of ID3 mRNA in endometrial explants. The expressions of ID2 and ID4 mRNAs were higher in endometria from gilts with somatic cell nuclear transfer-derived conceptuses compared with endometria from gilts carrying conceptuses derived from natural mating on Day 12. These results indicate that the expressions of ID family genes are dynamically regulated at the maternal-conceptus interface, suggesting that ID proteins may play critical roles in the regulation of endometrial epithelial cell function and conceptus development to support the establishment and maintenance of pregnancy in pigs.

ID1 contributes to cell growth invasion and migration in salivary adenoid cystic carcinoma.

Previous studies have reported that inhibitor of DNA binding 1 (ID1) exerts an oncogenic role in a number of tumors. In the present study, the role of ID1 in the growth, invasion and migration of salivary adenoid cystic carcinoma (SACC) cells was investigated. ID1 expression in clinical SACC samples was compared with that in normal salivary tissues using immunohistochemical staining, and the correlation between ID1 expression and clinical pathological characteristics was then determined. Subsequently, ID1 was overexpressed or silenced to investigate the effects of ID1 expression on SACC cell proliferation, invasion and migration. In addition, the gene expression levels of known ID1 target genes, including S100A9, CDKN2A and matrix metalloproteinase 1 (MMP1) was measured using reverse transcription-quantitative polymerase chain reaction to elucidate the potential mechanisms of ID1 in SACC. The results of the present study indicated that the protein expression levels of ID1 were significantly increased in the SACC tissues compared with that in the normal salivary tissues (P<0.001), and a positive correlation between ID1 expression and tumor stage (P=0.001), tumor invasion (P=0.002) and metastasis (P=0.019) in SACC was observed. Knockdown of ID1 in SACC cells significantly inhibited cell growth, invasion and migration (all P<0.01), whereas overexpression of ID1 promoted cell proliferation, invasion and migration (all P<0.01). The gene expression level of MMP1 was significantly reduced following ID1 knockdown in SACC-83 cells when compared with negative controls (P<0.05), whereas S100A9 and CDKN2A expression levels were significantly upregulated (both P<0.05). The results suggest that ID1 may regulate the growth, invasion and migration of SACC cells, and that MMP1, S100A9 and CDKN2A may serve as target genes of ID1 and mediate the effects of ID1 in SACC cells. Therefore, ID1 may present a potential target gene for the treatment of patients with SACC to inhibit cancer cell growth and metastasis.

RUNX3 plays an important role in mediating the BMP9-induced osteogenic differentiation of mesenchymal stem cells.

Although bone morphogenetic protein9(BMP9) is highly capable of promoting the osteogenic differentiation of mesenchymal stem cells(MSCs) both invitro and invivo, the molecular mechanisms involved remain to be fully elucidated. Runt-related transcription factor(RUNX)3 is an essential regulator of osteoblast/chondrocyte maturation. However, the exact role of RUNX3 in BMP9 osteoinductive activity is unknown. In this study, we sought to investigate the functional role of RUNX3 in the BMP9-induced osteogenic differentiation of MSCs. We found that BMP9 upregulated the endogenous expression of RUNX3 in MSCs. The overexpression or/and knockdown of RUNX3 both increased the levels of alkaline phosphatase(ALP) a marker of BMP9-induced early osteogenic differentiation. Nevertheless, matrix mineralization, a marker of BMP9-induced late osteogenic differentiation was enhanced by the overexpression of RUNX3, whereas it was inhibited by the knockdown of RUNX3. The BMP9-induced expression of osteogenic pivotal transcription factors[inhibitor of differentiation(Id)3, distal-less homeobox5(DLX5) and RUNX2)] was further increased by the overexpression of RUNX3; however, it was reduced by the knockdown of RUNX3. However, the expression levels of Id1 and Id2 were both enhanced by the overexpression or/and knockdown of RUNX3. The BMP9-induced phosphorylation of Smad1/5/8 was increased with the overexpression of RUNX3, and yet was decreased with the knockdown of RUNX3. Collectively, our findings suggest that RUNX3 is an essential modulator of the BMP9-induced osteoblast lineage differentiation of MSCs.

RNA sequencing of bipolar disorder lymphoblastoid cell lines implicates the neurotrophic factor HRP-3 in lithium’s clinical efficacy

Objectives: Lithium remains the oldest and most effective treatment for mood stabilisation in bipolar disorder (BD), even though at least half of patients are only partially responsive or do not respond. This study aimed to identify biomarkers associated with lithium response in BD, based on comparing RNA sequencing information derived from lymphoblastoid cell lines (LCLs) of lithium-responsive (LR) versus lithium non-responsive (LNR) BD patients, to assess gene expression variations that might bear on treatment outcome.Methods: RNA sequencing was carried out on 24 LCLs from female BD patients (12 LR and 12 LNR) followed by qPCR validation in two additional independent cohorts (41 and 17 BD patients, respectively).Results: Fifty-six genes showed nominal differential expression comparing LR and LNR (FC ≥ |1.3|, P ≤ 0.01). The differential expression of HDGFRP3 and ID2 was validated by qPCR in the independent cohorts.Conclusions: We observed higher expression levels of HDGFRP3 and ID2 in BD patients who favourably respond to lithium. Both of these genes are involved in neurogenesis, and HDGFRP3 has been suggested to be a neurotrophic factor. Additional studies in larger BD cohorts are needed to confirm the potential of HDGFRP3 and ID2 expression levels in blood cells as tentative favourable lithium response biomarkers.

Retinoic acid regulates avian lung branching through a molecular network.

Retinoic acid (RA) is of major importance during vertebrate embryonic development and its levels need to be strictly regulated otherwise congenital malformations will develop. Through the action of specific nuclear receptors, named RAR/RXR, RA regulates the expression of genes that eventually influence proliferation and tissue patterning. RA has been described as crucial for different stages of mammalian lung morphogenesis, and as part of a complex molecular network that contributes to precise organogenesis; nonetheless, nothing is known about its role in avian lung development. The current report characterizes, for the first time, the expression pattern of RA signaling members (stra6, raldh2, raldh3, cyp26a1, rarα, and rarβ) and potential RA downstream targets (sox2, sox9, meis1, meis2, tgfβ2, and id2) by in situ hybridization. In the attempt of unveiling the role of RA in chick lung branching, in vitro lung explants were performed. Supplementation studies revealed that RA stimulates lung branching in a dose-dependent manner. Moreover, the expression levels of cyp26a1, sox2, sox9, rarβ, meis2, hoxb5, tgfβ2, id2, fgf10, fgfr2, and shh were evaluated after RA treatment to disclose a putative molecular network underlying RA effect. In situ hybridization analysis showed that RA is able to alter cyp26a1, sox9, tgfβ2, and id2 spatial distribution; to increase rarβ, meis2, and hoxb5 expression levels; and has a very modest effect on sox2, fgf10, fgfr2, and shh expression levels. Overall, these findings support a role for RA in the proximal-distal patterning and branching morphogenesis of the avian lung and reveal intricate molecular interactions that ultimately orchestrate branching morphogenesis.