Comparative efficiency of differential diagnostic methods for the identification of BRAF V600E gene mutation in papillary thyroid cancer (Review).

V-Raf murine sarcoma viral oncogene homolog B1 (BRAF) encodes a serine-threonine kinase. The V600E point mutation in the BRAF gene is the most common mutation, predominantly occurring in melanoma, and colorectal, thyroid and non-small cell lung cancer. Particularly in the context of thyroid cancer research, it is routinely employed as a molecular biomarker to assist in diagnosing and predicting the prognosis of papillary thyroid cancer (PTC), and to formulate targeted therapeutic strategies. Currently, several methods are utilized in clinical settings to detect BRAF V600E mutations in patients with PTC. However, the sensitivity and specificity of various detection techniques vary significantly, resulting in diverse detection outcomes. The present review highlights the advantages and disadvantages of the methods currently employed in medical practice, with the aim of guiding clinicians and researchers in selecting the most suitable detection approach for its high sensitivity, reproducibility and potential to develop targeted therapeutic regimens for patients with BRAF gene mutation-associated PTC.

Construction of an immune-related lncRNA-miRNA-mRNA regulatory network in radiation-induced esophageal injury in rats.

Radiation-induced esophageal injury (RIEI) is an adverse reaction of radiation therapy in patients with esophageal cancer, lung cancer and other malignant tumors. Competitive endogenous RNA (ceRNA) network is known to play a significant role in the onset and progression of many diseases, but the exact mechanism of ceRNA in RIEI has not been fully elucidated. In this study, rat esophaguses were obtained after conducting irradiation under different doses (0 Gy, 25 Gy, 35 Gy). Total RNA was extracted and mRNA, lncRNA, circRNA, and miRNA sequencing was performed. Multiple dose-dependent differentially expressed RNAs (dd-DERs), including 870 lncRNAs, 82 miRNAs, 2478 mRNAs, were obtained through the integration of differential expression analysis and dose-dependent screening (35 Gy ≥ 25 Gy > 0 Gy, or 35 Gy ≤ 25 Gy < 0 Gy). Co-expression analysis and prediction of the binding site in dd-DER were conducted and 27 lncRNAs, 20 miRNAs, and 168 mRNAs were selected to construct a ceRNA network. As the immune microenvironment is crucial for RIEI progression, we constructed an immune-related ceRNA network consisting of 11 lncRNAs, 9 miRNAs, and 9 mRNAs. The expression levels of these immune-related RNAs were verified by RT-qPCR. Immune infiltration analysis showed that the RNAs in the immune-related ceRNA network were mainly associated with the proportion of monocytes, M2 macrophages, activated NK cells, and activated CD4+ memory T cells. Drug sensitivity analysis was conducted based on the expression levels of mRNAs in the immune-related ceRNA network, and small molecule drugs with preventive and therapeutic effects on RIEI were identified. In summary, an immune-related ceRNA network associated with RIEI progression was constructed in this study. The findings provide useful information on new potential targets for the prevention and treatment of RIEI.

N6-methyladenosine-mediated LDHA induction potentiates chemoresistance of colorectal cancer cells through metabolic reprogramming.

Background: Chemoresistance to 5-fluorouracil (5-FU) is a major barrier to influence the treatment efficiency of colorectal cancer (CRC) patients, while the precise molecular mechanisms underlying 5-FU resistance remain to be fully elucidated. Methods: The metabolic profiles including ATP generation, glucose consumption, lactate generation, and oxygen consumption rate (OCR) in 5-FU resistant CRC cells were compared with those in their parental cells. Subsequently, a series of in vitro and in vivo experiments were carried out to investigate the mechanisms responsible for metabolic reprogramming of 5-FU resistant CRC cells. Results: We found that 5-FU resistant CRC cells showed increased levels of ATP generation, glucose consumption, lactate generation, and OCR as compared with those in their parental cells. Further, increased levels of mRNA N6-methyladenosine (m6A) and methyltransferase-like 3 (METTL3) were observed in 5-FU resistant CRC cells. Inhibition or knockdown of METTL3 can suppress glycolysis and restore chemosensitivity of 5-FU resistant CRC cells. Mechanistically, METTL3 enhances the expression of LDHA, which catalyzes the conversion of pyruvate to lactate, to trigger glycolysis and 5-FU resistance. METTL3 can increase the transcription of LDHA via stabilizing mRNA of hypoxia-inducible factor (HIF-1α), further, METTL3 also triggers the translation of LDHA mRNA via methylation of its CDS region and recruitment of YTH domain-containing family protein 1 (YTHDF1). Targeted inhibition of METTL3/LDHA axis can significantly increase the in vitro and in vivo 5-FU sensitivity of CRC cells. Conclusion: Our study indicates that METTL3/LDHA axis-induced glucose metabolism is a potential therapy target to overcome 5-FU resistance in CRC cells.

Construction of a Tumor Immune Microenvironment-Related Prognostic Model in BRAF-Mutated Papillary Thyroid Cancer.

BRAF mutation is a representative oncogenic mutation, with a frequency of 60% in papillary thyroid carcinoma (PTC), but the reasons for the poor prognosis and more aggressive course of BRAF-mutated PTC are controversial. Tumor immune microenvironment (TIME) is an essential factor permitting the development and progression of malignancy, but whether TIME participates in the prognosis of BRAF-mutated PTC has not yet been reported. The primary goal of the present study was to provide a comprehensive TIME-related prognostic model to increase the predictive accuracy of progression-free survival (PFS) in patients with BRAF-mutated PTC. In this study, we analyzed the mRNA-seq data and corresponding clinical data of PTC patients obtained from the TCGA database. By calculating the TIME scores (immune score, stromal score and ESTIMATE score), the BRAF mutation group (n=237) was dichotomized into the high- and low-score groups. By functional analysis of differentially expressed genes (DEGs) in different high/low score groups, we identified 2 key TIME-related genes, HTR3A and NIPAL4, which affected PFS in BRAF-mutated PTC. A risk scoring system was developed by multivariate Cox analysis based on the abovementioned 2 TIME-related genes. Then, the BRAF-mutated cohort was divided into the high- and low-risk groups using the median risk score as a cutoff. A high risk score correlated positively with a higher HTR3A/NIPAL4 expression level but negatively with PFS in BRAF-mutated PTC. Ultimately, a nomogram was constructed by combining risk score with clinical parameter (Tumor stage), and the areas under the ROC curve (AUCs) of the nomogram for predicting 1-, 3- and 5-year PFS were then calculated and found to be 0.694, 0.707 and 0.738, respectively, indicating the improved accuracy and clinical utility of the nomogram versus the risk score model in the BRAF-mutated PTC cohort. Moreover, we determined the associations between prognostic genes or risk score and immune cell infiltration by two-way ANOVA. In the high-risk score, high HTR3A expression, and high NIPAL4 expression groups, higher infiltration of immune cells was found. Collectively, these findings confirm that the nomogram is effective in predicting the outcome of BRAF-mutated PTC and will add a spatial dimension to the developing risk stratification system.

Transcriptome Profiling Unveils a Critical Role of IL-17 Signaling-Mediated Inflammation in Radiation-Induced Esophageal Injury in Rats.

Elucidation of the molecular mechanisms involving the initiation and progression of radiation-induced esophageal injury (RIEI) is important for prevention and treatment. Despite ongoing advances, the underlying mechanisms controlling RIEI remain largely unknown. In the present study, RNA-seq was performed to characterize mRNA profiles of the irradiated rat esophagus exposed to 0, 25, or 35 Gy irradiation. Bioinformatics analyses including dose-dependent differentially expressed genes (DEGs), Gene Ontology (GO), Kyoto Encyclopedia of Gene and Genome (KEGG) pathway, protein-protein interaction (PPI) network, and immune infiltration were performed. 134 DEGs were screened out with a dose-dependent manner (35 Gy > 25 Gy > control, or 35 Gy < 25 Gy < control). GO and KEGG analyses showed that the most significant mechanism was IL-17 signaling-mediated inflammatory response. 5 hub genes, Ccl11, Cxcl3, Il17a, S100a8, and S100a9, were identified through the intersection of the DEGs involved in inflammatory response, IL-17 pathway, and PPI network. Additionally, immune infiltration analysis showed the activation of macrophages, monocytes, T cells, NKT cells, and neutrophils, among which macrophages, monocytes, and neutrophils might be the main sources of S100a8 and S100a9. Thus, these findings further our understanding on the molecular biology of RIEI and may help develop more effective therapeutic strategies.

Construction of a Signature Model to Predict the Radioactive Iodine Response of Papillary Thyroid Cancer.

Papillary thyroid cancer (PTC) accounts for about 90% of thyroid cancer. There are approximately 20%-30% of PTC patients showing disease persistence/recurrence and resistance to radioactive iodine (RAI) treatment. For these PTC patients with RAI refractoriness, the prognosis is poor. In this study, we aimed to establish a comprehensive prognostic model covering multiple signatures to increase the predictive accuracy for progression-free survival (PFS) of PTC patients with RAI treatment. The expression profiles of mRNAs and miRNAs as well as the clinical information of PTC patients were extracted from TCGA and GEO databases. A series of bioinformatics methods were successfully applied to filtrate a two-RNA model (IPCEF1 and hsa-mir-486-5p) associated with the prognosis of RAI-therapy. Finally, the RNA-based risk score was calculated based on the Cox coefficient of the individual RNA, which achieved good performances by the time-dependent receiver operating characteristic (tROC) curve and PFS analyses. Furthermore, the predictive power of the nomogram, integrated with the risk score and clinical parameters (age at diagnosis and tumor stage), was assessed by tROC curves. Collectively, our study demonstrated high precision in predicting the RAI response of PTC patients.

Metabolic Profiling Implicates a Critical Role of Cyclooxygenase-2-Mediated Arachidonic Acid Metabolism in Radiation-Induced Esophageal Injury in Rats.

Radiation-induced esophageal injury (RIEI) is a major dose-limiting complication of radiotherapy, especially for esophageal and thoracic cancers. RIEI is a multi-factorial and multi-step process, which is regulated by a complex network of DNA, RNA, protein and metabolite. However, it is unclear which esophageal metabolites are altered by ionizing radiation and how these changes affect RIEI progression. In this work, we established a rat model of RIEI with 0-40 Gy X-ray irradiation. Esophageal irradiation using ≥25 Gy induced significant changes to rats, such as body weight, food intake, water intake and esophageal structure. The metabolic changes and related pathways of rat esophageal metabolites were investigated by liquid chromatography-mass spectrometry (LC-MS). One hundred eighty metabolites showed an up-regulation in a dose-dependent manner (35 Gy ≥ 25 Gy > controls), and 199 metabolites were downregulated with increasing radiation dose (35 Gy ≤ 25 Gy < controls). The KEGG analysis showed that ionizing radiation seriously disrupted multiple metabolic pathways, and arachidonic acid metabolism was the most significantly enriched pathway. 20 metabolites were dysregulated in arachidonic acid metabolism, including up-regulation of five prostaglandins (PGA2, PGJ2, PGD2, PGH2, and PGI2) in 25 or 35 Gy groups. Cyclooxygenase-2 (COX-2), the key enzyme in catalyzing the biosynthesis of prostaglandins from arachidonic acid, was highly expressed in the esophagus of irradiated rats. Additionally, receiver operating characteristic (ROC) curve analysis revealed that PGJ2 may serve as a promising tissue biomarker for RIEI diagnosis. Taken together, these findings indicate that ionizing radiation induces esophageal metabolic alterations, which advance our understanding of the pathophysiology of RIEI from the perspective of metabolism.

Serum Metabolomic Analysis of Radiation-Induced Lung Injury in Rats.

Radiation-induced lung injury is a common complication of radiotherapy for lung cancer, breast cancer, esophageal cancer, and thymoma. This study aims to illustrate biomarkers of radiation-induced lung injury and its potential mechanism through the study of metabolomic alterations in serum of Sprague-Dawley rats with different radiation doses. Serum from 0, 10, or 20 Gy irradiated rats were collected and subjected to gas chromatography-mass spectrometry. The result showed that there were 23 dysregulated metabolites between the 10 Gy irradiation group and the 0 Gy control group, whereas 36 preferential metabolites were found between the 20 Gy irradiated rat serum and the control groups. Among them, there were 19 common differential metabolites in the 2 irradiation groups, including 3 downregulated (benzyl thiocyanate, carbazole, and N-formyl-L-methionine) and 16 upregulated metabolites. We further analyzed the metabolic pathways of different metabolites; the results showed that there were 3 significant enrichment pathways in the 10 Gy vs 0 Gy group and 7 significant enrichment pathways in the 20 Gy vs 0 Gy group. Among them, taurine and hypotaurine metabolism, riboflavin metabolism, and glyoxylate and dicarboxylate metabolism were the common metabolic enrichment pathways of the 10 Gy vs 0 Gy group and the 20 Gy vs 0 Gy group.

Effect of kaempferol on hedgehog signaling pathway in rats with --chronic atrophic gastritis - Based on network pharmacological screening and experimental verification.

OBJECTIVE: The effect of active ingredients of Chaishaoliujun Decoction (CD) on chronic atrophic gastritis (CAG) was screened by network pharmacological method and verified by preliminary experiment. METHODS: Firstly, the active ingredients and drug targets of CD were retrieved in TCMSP database; CAG-related targets from PharmGkb, OMIM, GeneCards and DrugBank databases were collected as well. Secondly, the drug targets and disease targets were mapped to obtain the intersection targets. PPI network and active ingredient-common target network were constructed for the intersection targets obtained and KEGG enrichment analysis was also carried out. Finally, the core active ingredient (kaempferol), effective targets (IL-1ß、IL-6) and hedgehog signaling pathway were verified by animal experiments. RESULTS: There were 137 active ingredients, 243 potential target so and 48 intersection targets with CAG in CD. 147 KEGG enrichment pathways were obtained, mainly involving JAK/STAT signaling pathway, PI3K/Akt signaling pathway, hedgehog signaling pathway, etc. The results of animal experiments showed: The content of IL-1ß and IL-6 in model group was significantly increased compared with the normal group, while the mRNA and protein expressions of Shh, Ptch1 and Gli1 were also significantly decreased (P < 0.05); compared with model group, the content of IL-1ß and IL-6 in the vitacoenzyme group, the CD group and the kaempferol group were significantly decreased, while the mRNA and protein expressions of Shh, Ptch1 and Gli1 were significantly increased (P < 0.05). CONCLUSION: Kaempferol, the active ingredient of CD, could reduce the levels of IL-6 and IL-1ß by regulating hedgehog signaling pathway so as to play a role in the treatment of CAG. Hence this paper could provide the methodological basis and theoretical basis for further revealing the pharmacological mechanism of CD.

Integrative multi-omic analysis of radiation-induced skin injury reveals the alteration of fatty acid metabolism in early response of ionizing radiation.

BACKGROUND: Radiation-induced skin injury is a serious concern during radiotherapy and accidental exposure to radiation. OBJECTIVE: This study aims to investigate the molecular events in early response to ionizing radiation of skin tissues and underlying mechanism. METHODS: Mice and rats were irradiated with an electron beam. Skin tissues were used for liquid chromatography-mass spectrometry (LC-MS)-based metabolomics, mRNA-Seq and single-cell RNA sequencing (scRNA-Seq). Human keratinocytes (HaCaT) and skin fibroblasts (WS1) were used for functional studies. RESULTS: The integrated analysis of metabolomics and transcriptomics showed that 6 key fatty acid-associated metabolites, 9 key fatty acid-associated genes and multiple fatty acid-associated pathways were most obviously enriched and increased in the irradiated skins. Among them, acyl-CoA dehydrogenase very long chain (ACADVL) was investigated in greater detail due to its most obvious expression difference and significance in fatty acid metabolism. ScRNA-Seq of rat skin from irradiated individuals revealed that ACADVL was expressed in all subpopulations of skin tissues, with variations at different timepoints after radiation. Immunohistochemistry confirmed an increased ACADVL expression in the epidermis from human sample and various animal models, including monkeys, rats and mice. The knockdown of ACADVL increased the radiosensitivity of human keratinocytes and human skin fibroblasts. Silencing of ACADVL facilitated the expression of apoptosis and pyroptosis-related proteins following ionizing radiation. CONCLUSION: This study illustrated that cutaneous fatty acid metabolism was altered in the early response of ionizing radiation, and fatty acid metabolism-associated ACADVL is involved in radiation-induced cell death.

Radiotherapy-Induced Digestive Injury: Diagnosis, Treatment and Mechanisms.

Radiotherapy is one of the main therapeutic methods for treating cancer. The digestive system consists of the gastrointestinal tract and the accessory organs of digestion (the tongue, salivary glands, pancreas, liver and gallbladder). The digestive system is easily impaired during radiotherapy, especially in thoracic and abdominal radiotherapy. In this review, we introduce the physical classification, basic pathogenesis, clinical characteristics, predictive/diagnostic factors, and possible treatment targets of radiotherapy-induced digestive injury. Radiotherapy-induced digestive injury complies with the dose-volume effect and has a radiation-based organ correlation. Computed tomography (CT), MRI (magnetic resonance imaging), ultrasound (US) and endoscopy can help diagnose and evaluate the radiation-induced lesion level. The latest treatment approaches include improvement in radiotherapy (such as shielding, hydrogel spacers and dose distribution), stem cell transplantation and drug administration. Gut microbiota modulation may become a novel approach to relieving radiogenic gastrointestinal syndrome. Finally, we summarized the possible mechanisms involved in treatment, but they remain varied. Radionuclide-labeled targeting molecules (RLTMs) are promising for more precise radiotherapy. These advances contribute to our understanding of the assessment and treatment of radiation-induced digestive injury.

Construction of a Novel Gene-Based Model for Survival Prediction of Hepatitis B Virus Carriers With HCC Development.

Despite the effectiveness of hepatitis B virus (HBV) vaccination in reducing the prevalence of chronic HBV infection as well as the incidence of acute hepatitis B, fulminant hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC), there was still a large crowd of chronically infected populations at risk of developing cirrhosis or HCC. In this study, we established a comprehensive prognostic system covering multiple signatures to elevate the predictive accuracy for overall survival (OS) of hepatitis B virus carriers with HCC development. Weighted Gene Co-Expression Network Analysis (WGCNA), Least Absolute Shrinkage and Selection Operator (LASSO), Support Vector Machine Recursive Feature Elimination (SVM-RFE), and multivariate COX analysis, along with a suite of other online analyses were successfully applied to filtrate a three-gene signature model (TP53, CFL1, and UBA1). Afterward, the gene-based risk score was calculated based on the Cox coefficient of the individual gene, and the prognostic power was assessed by time-dependent receiver operating characteristic (tROC) and Kaplan-Meier (KM) survival analysis. Furthermore, the predictive power of the nomogram, integrated with the risk score and clinical parameters (age at diagnosis and TNM stage), was revealed by the calibration plot and tROC curves, which was verified in the validation set. Taken together, our study may be more effective in guiding the clinical decision-making of personalized treatment for HBV carriers.

Activated Beta-Catenin Signaling Ameliorates Radiation-Induced Skin Injury by Suppressing Marvel D3 Expression.

Radiation-induced skin injury remains a serious concern for cancer radiotherapy, radiation accidents and occupational exposure, and the damage mainly occurs due to apoptosis and reactive oxygen species (ROS) generation. There is currently no effective treatment for this disorder. The ß-catenin signaling pathway is involved in the repair and regeneration of injured tissues. However, the role of the ß-catenin signaling pathway in radiation-induced skin injury has not been reported. In this study, we demonstrated that the ß-catenin signaling pathway was activated in response to radiation and that its activation by Wnt3a, a ligand-protein involved in the ß-catenin signaling pathway, inhibited apoptosis and the production of ROS in irradiated human keratinocyte HaCaT cells and skin fibroblast WS1 cells. Additionally, Wnt3a promoted cell migration after irradiation. In a mouse model of full-thickness skin wounds combined with total-body irradiation, Wnt3a was shown to facilitate skin wound healing. The results from RNA-Seq revealed that 24 genes were upregulated and 154 were downregulated in Wnt3a-treated irradiated skin cells, and these dysregulated genes were mainly enriched in the tight junction pathway. Among them, Marvel D3 showed the most obvious difference. We further found that the activated ß-catenin signaling pathway stimulated the phosphorylation of JNK by silencing Marvel D3. Treatment of irradiated cells with SP600125, a JNK inhibitor, augmented ROS production and impeded cell migration. Furthermore, treatment with Wnt3a or transfection with Marvel D3-specific siRNAs could reverse the above effects. Taken together, these findings illustrate that activated ß-catenin signaling stimulates the activation of JNK by negatively regulating Marvel D3 to ameliorate radiation-induced skin injury.

Testis-specific protein, Y-linked 1 activates PI3K/AKT and RAS signaling pathways through suppressing IGFBP3 expression during tumor progression.

The testis-specific protein, Y-linked 1 (TSPY1), a newly recognized cancer/testis antigen, has been suggested to accelerate tumor progression. However, the mechanisms underlying TSPY1 cancer-related function remain limited. By mining the RNA sequencing data of lung and liver tumors from The Cancer Genome Atlas, we found frequent ectopic expression of TSPY1 in lung adenocarcinoma (LUAD) and liver hepatocellular carcinoma (LIHC), and the male-specific protein was associated with higher mortality rate and worse overall survival in patients with LUAD and LIHC. Overexpression of TSPY1 promotes cell proliferation, invasiveness, and cycle transition and inhibits apoptosis, whereas TSPY1 knockdown has the opposite effects on these cancer cell phenotypes. Transcriptomic analysis revealed the involvement of TSPY1 in PI3K/AKT and RAS signaling pathways in both LUAD and LIHC cells, which was further confirmed by the increase in the levels of phosphorylated proteins in the PI3K-AKT and RAS signaling pathways in TSPY1-overexpressing cancer cells, and by the suppression on the activity of these two pathways in TSPY1-knockdown cells. Further investigation identified that TSPY1 could directly bind to the promoter of insulin growth factor binding protein 3 (IGFBP3) to inhibit IGFBP3 expression and that downregulation of IGFBP3 increased the activity of PI3K/AKT/mTOR/BCL2 and RAS/RAF/MEK/ERK/JUN signaling in LUAD and LIHC cells. Taken together, the observations reveal a novel mechanism by which TSPY1 could contribute to the progression of LUAD and LIHC. Our finding is of importance for evaluating the potential of TSPY1 in immunotherapy of male tumor patients with TSPY1 expression.

TSPY1 suppresses USP7-mediated p53 function and promotes spermatogonial proliferation.

Testis-specific protein Y-linked 1 (TSPY1) is expressed predominantly in adult human spermatogonia and functions in the process of spermatogenesis; however, our understanding of the underlying mechanism is limited. Here we observed that TSPY1, as an interacting partner of TSPY-like 5 (TSPYL5), enhanced the competitive binding of TSPYL5 to ubiquitin-specific peptidase 7 (USP7) in conjunction with p53. This activity, together with its promotion of TSPYL5 expression by acting as a transcription factor, resulted in increased p53 ubiquitylation. Moreover, TSPY1 could decrease the p53 level by inducing the degradation of ubiquitinated USP7. We demonstrated that the promotion of p53 degradation by TSPY1 influenced the activity of p53 target molecules (CDK1, p21, and BAX) to expedite the G2/M phase transition and decrease cell apoptosis, accelerating cell proliferation. Taken together, the observations reveal the significance of TSPY1 as a suppressor of USP7-mediated p53 function in inhibiting p53-dependent cell proliferation arrest. By simulating TSPY1 function in Tspy1-deficient spermatogonia derived from mouse testes, we found that TSPY1 could promote spermatogonial proliferation by decreasing the Usp7-modulated p53 level. The findings suggest an additional mechanism underlying the regulation of spermatogonial p53 function, indicating the significance of TSPY1 in germline homeostasis maintenance and the potential of TSPY1 in regulating human spermatogonial proliferation via the USP7-mediated p53 signaling pathway.

Epigenetic modifications promote the expression of the orphan nuclear receptor NR0B1 in human lung adenocarcinoma cells.

The ectopic activation of NR0B1 is involved in the development of some cancers. However, the regulatory mechanisms controlling NR0B1 expression are not well understood. Therefore, the epigenetic modifications promoting NR0B1 activation were examined in this study. NR0B1 protein was detected in cancerous tissues of more than 50% of human lung adenocarcinoma (ADCA) cases and tended to be expressed in low-differentiated cancerous tissues obtained from males. Nevertheless, NR0B1 activation in ADCA has not previously been correlated with DNA demethylation. NR0B1 expression was not detected in 293T cells, although it contains a hypomethylated NR0B1 promoter. Treating 293T cells with a histone deacetylase inhibitor increased acetylated histone H4 binding to the NR0B1 promoter and activated NR0B1 expression. In contrast, treatment with histone methylase inhibitors decreased the methylation of histones H3K9 and H3K27 and slightly induced NR0B1 transcription. Furthermore, the level of acetyl-histone H4 binding to the NR0B1 promoter increased, whereas the occupancy of H3K27me3 was lower in cancerous tissues than in non-cancerous tissues. Similar histone occupancies were confirmed in a comparison of cancerous tissues with strong, moderate and negative NR0B1 expression. In conclusion, this study shows that CpG methylation within the NR0B1 promoter is not involved in the in vivo regulation of NR0B1 expression, whereas the hyperacetylation of histone H4 and the unmethylation of histones H3K9 and H3K27, and their binding to the NR0B1 promoter results in decondensed euchromatin for NR0B1 activation.