BACKGROUND & AIMS:CD8+ T cells play a crucial role in antiviral immunity; however, hepatitis B virus (HBV)-specific CD8+ T cells become dysfunctional during chronic HBV (CHB) infection. Blocking inhibitory pathways only partially restores efficient antiviral responses, suggesting that the mechanism underlying CD8+ T-cell dysfunction is complicated. This study aimed to investigate whether HBV-specific CD8+ T cells undergo ferroptosis, examine its correlation with T-cell dysfunction, and elucidate the underlying mechanism and potential intervention strategies. METHODS:Analysis of CD8+ T cells from patients with CHB revealed ferroptosis markers via flow cytometry, electron microscopy, and single-cell RNA sequencing, and HBV-specific CD8+ T cells were identified using HBV-core antigen peptide-loaded major histocompatibility complex I tetramer. Flow cytometry, single-cell RNA sequencing, and additional experimental approaches were employed to investigate the ferroptosis-associated mechanisms in CD8+ T-cell dysfunction. RESULTS:Ferroptosis was observed in CD8+ T cells from patients with CHB, as indicated with increased lipid peroxidation, Fe2+ accumulation and mitochondrial atrophy, particular in HBV-specific CD8+ T cells. Downregulation of glutathione peroxidase 4 and upregulation of CD36 were found in CD8+ T cells with high level of lipid peroxidation. Furthermore, ferroptosis is accompanied by impaired antiviral ability. Mechanistically, palmitic acid upregulates CD36 expression, leading to lipid accumulation, whereas elevated serum transforming growth factor beta 1 in patients with CHB reduces GPX4 expression and impairs antioxidant capacity. These factors synergistically promote ferroptosis and dysfunction of CD8+ T cells. Treatment with selenium and liproxstain-1 enhanced cytotoxic cytokines production by CD8+ T cells, and subsequently improved antiviral efficacy in vivo. CONCLUSIONS:Our study demonstrates that ferroptosis drives CD8+ T-cell dysfunction in chronic HBV infection. Targeting ferroptosis pathways may offer a novel therapeutic strategy to restore antiviral immunity.

BackgroundImmunotherapy has recently become a hot topic in the field of oncology, with PD-L1 playing a crucial role in this area. However, the research on PD-L1 correlation prediction models is not fully understood. The aim of our study was to investigate the role of PD-L1-related genes in lung adenocarcinoma immunity.MethodsThe mRNA and clinical data were obtained from the Cancer Genome Atlas database. DESeq2, Glmnet, forestplot, clusterProfiler and enrichplot were used to analyze the mRNA and clinical data. Western blotting and real-time qRT-PCR were used to confirm the GPR115, MF12, GREB1L, SPRR1B and LIPK mRNA and protein expression.ResultsFirstly, 562 cases of TCGA lung adenocarcinoma, including 503 of tumor tissue and 59 of normal tissue were collected. The dataset was analyzed using the DESeq2 package of R. 1,251 high- and 285 low-expression genes were obtained. The tumor samples were divided into CD274-high and CD274-low expression samples and 873 genes were up-regulated and 1,010 genes were down regulated between CD274-high and CD274-low samples. Subsequently, the intersection of 1,251 and 873 was taken to obtain 110 genes that were both highly expressed genes in tumors and CD274 high-expression samples. Survival analysis of 110 genes yielded 5 meaningful genes including GPR115, MF12, GREB1L, SPRR1B, and LIPK (p < 0.001). These five genes were used to construct PD-L1 risk predictors. Cytokine-cytokine receptor interaction and IL-17 signaling pathway were involved in the regulation of this risk model factors to lung adenocarcinoma. The level of effector memory CD4 T cells and Type 2 T helper cells were correlated with the risk model factor. Importantly, the PD-L1 risk prediction model could effectively predict the prognosis of patients.ConclusionThe construction of PD-L1 risk model was of great significance for the treatment of lung adenocarcinoma.

Background: Osteosarcoma is the most common type of primary malignant bone tumor. Introduction: This study aimed to explore potential key prognostic genes and their roles in osteosarcoma. Methods: Three microarray datasets for osteosarcoma were downloaded from the GEO database. Differentially expressed genes (DEGs) were screened by the Limma package. Functional enrichment analysis was performed based on DAVID, GeneMANIA, and Metascape databases. Prognostic value of DEGs was elevated by survival analysis. CIBERSORT was used to assess the infiltrating abundance of 22 immune cells, followed by the Pearson correlation analysis between immune cells and prognosis-related genes. Gene set enrichment analysis and drug-gene interactions prediction were performed for prognosis-related genes. Results: A total of 8 common up-regulated DEGs and 13 common down-regulated DEGs were screened in the GSE36001 and GSE56001 datasets. Enrichment analysis showed these DEGs were implicated in platelet activation, SMAD protein phosphorylation, lymphocyte/leukocyte/T cells activation, and cell migration. Survival analysis indicated that elevated expression of ADAM19 and TUBB1 were associated with a favorable prognosis. CIBERSORT algorithm revealed the higher infiltrating level of CD8 T cells, macrophages M0, and M2 in osteosarcoma. ADAM19 expression positively correlated with naïve B cells and negatively correlated with activated dendritic cells infiltrating abundance. TUBB1 expression positively correlated with gamma delta T cells while negatively correlated with helper follicular T cells infiltrating abundance. A total of 56 drugs were found to target TUBB1. Conclusion: ADAM19 and TUBB1 could be prognostic biomarkers in osteosarcoma. Both their expression correlates with tumor infiltrating immune cells. TUBB1 was a multi-drug target that might be a therapeutic target in osteosarcoma.

Objective: The function of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) has been studied in several cancers; however, little is known about its role in mantle cell lymphoma (MCL). Thus, this study aimed to characterize the significance and function of EHMT2 in MCL. Methods: EHMT2 expression in MCL and reactive hyperplasia (RH) were investigated by immunohistochemistry. Genome-wide analysis of DNA methylation was performed on EHMT2 + MCL samples. The function of EHMT2 was determined by CCK8, flow cytometry, and western blot assays. Gene expression profile analysis was performed before and after EHMT2 knockdown to search for EHMT2-regulated genes. Co-immunoprecipitation (Co-IP) experiments were conducted to identify the proteins interacting with EHMT2. Results: EHMT2 was expressed in 68.57% (24/35) of MCLs but not in any RHs. Genome-wide analysis of DNA methylation on EHMT2 + MCLs revealed that multiple members of the HOX, FOX, PAX, SOX, and CDX families were hypermethylated or hypomethylated in EHMT2 + MCLs. BIX01294, a EHMT2 inhibitor, inhibited MCL cell growth and stalled cells in the G1 phase. Additionally, BIX01294 downregulated the expressions of cell cycle proteins, cyclin D1, CDK4, and P21, but upregulated the expressions of apoptosis-related proteins, Bax and caspase-3. Co-IP experiments revealed that EHMT2 interacted with UHRF1, HDAC1, and HDAC2 but not with HDCA3. After EHMT2 knockdown, multiple genes were regulated, including CD5 and CCND1, mostly enriched in the Tec kinase signaling pathway. In addition, several genes (e.g., MARCH1, CCDC50, HIP1, and WNT3) were aberrantly methylated in EHMT2 + MCLs. Conclusions: For the first time, we determined the significance of EHMT2 in MCL and identified potential EHMT2-regulated genes.

Sarcomas are a heterogeneous group of malignant mesenchymal neoplasms. This study aimed to investigate the immune-related prognostic gene signatures in the tumor microenvironment of sarcoma. The RNA-sequencing data and clinical phenotype data of 260 sarcoma samples and two normal samples were downloaded from The Cancer Genome Atla (TCGA) database. Tumor purity and immune cells infiltration were evaluated by Estimation of Stromal and Immune cells in Malignant Tumors using Expression data (ESTIMATE) deconvolution algorithm. Differentially expressed genes (DEGs) were screened in high vs. low immune score groups. Survival analysis was performed using Kaplan-Meier curve with log-rank test. Tumor infiltrating of immune cells was analyzed by Tumor Immune Estimation Resource (TIMER). High immune score and ESTIMATE score were associated with favorable prognosis. A total of 623 immune DEGs were screened. The majority of these genes (532 genes accounting for 85% of the DEGs) were up-regulated, and these genes were significantly enriched in various immune related biological processed and pathways, such as neutrophil activation, T cell activation, antigen processing and presentation. A total of 146 prognosis-related immune DEGs, and seven hub genes were identified, including B2M, HLA-DRB1, HLA-DRA, HLA-E, LCK, HLA-DPA1, and VAV1. Survival analysis showed that high expression of these genes was associated with a favorable prognosis. There were negative correlations between the expression of these hub genes and tumor purity, while positive correlations between expression of these hub genes and f infiltration levels of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages and dendritic cells. These results help to stratify patients with different immune subtypes and help to design immunotherapy strategies for these patients in sarcoma.

Abstract Protein arginine methyltransferase 5 (PRMT5) has previously been reported to be upregulated in many malignant tumors. This study investigated the significance of PRMT5 in endometrial carcinoma (EC) and explored its function in tumorigenesis. Immunohistochemistry was performed to evaluate PRMT5 expression in 62 EC and 66 endometrial hyperplasia samples. The functions of PRMT5 were investigated by cell counting kit‐8, plate colony formation, wound healing, and transwell and flow cytometry assays. Quantitative reverse transcription‐polymerase chain reaction and western blotting were used to measure the expression of PRMT5, changes in estrogen receptor α (ERα), and related functional proteins. Coimmunoprecipitation was performed to examine the interaction of PRMT5 with ERα and its coactivator steroid receptor coactivator‐1 (SRC1). Compared to endometrial hyperplasia tissue, PRMT5 was overexpressed in endometrioid adenocarcinoma (EAC) but not overexpressed in mucinous EC. The main expression pattern of PRMT5 in EAC was cytoplasmic. However, the positive cases of endometrial hyperplasia showed both cytoplasmic and nuclear positivity in the endometrial glands or were mainly positive in stromal cells. Knockdown of PRMT5 significantly inhibited the growth and migration ability of EAC cells and promoted their apoptosis by regulating cyclin D1, c‐myc, p53, and Bcl2 proteins. Furthermore, PRMT5 could form a complex with ERα and SRC1 to promote the expression of ERα. In conclusion, PRMT5 plays a significant role in the progression of EAC by interacting with ERα and impacting the cell cycle signaling pathways.

Background This study hoped to explore the potential biomarkers and associated metabolites during osteosarcoma (OS) progression based on bioinformatics integrated analysis. Methods Gene expression profiles of GSE28424, including 19 human OS cell lines (OS group) and 4 human normal long bone tissue samples (control group), were downloaded. The differentially expressed genes (DEGs) in OS vs. control were investigated. The enrichment investigation was performed based on DEGs, followed by protein–protein interaction network analysis. Then, the feature genes associated with OS were explored, followed by survival analysis to reveal prognostic genes. The qRT-PCR assay was performed to test the expression of these genes. Finally, the OS-associated metabolites and disease-metabolic network were further investigated. Results Totally, 357 DEGs were revealed between the OS vs. control groups. These DEGs, such as CXCL12, were mainly involved in functions like leukocyte migration. Then, totally, 38 feature genes were explored, of which 8 genes showed significant associations with the survival of patients. High expression of CXCL12, CEBPA, SPARCL1, CAT, TUBA1A, and ALDH1A1 was associated with longer survival time, while high expression of CFLAR and STC2 was associated with poor survival. Finally, a disease-metabolic network was constructed with 25 nodes including two disease-associated metabolites cyclophosphamide and bisphenol A (BPA). BPA showed interactions with multiple prognosis-related genes, such as CXCL12 and STC2. Conclusion We identified 8 prognosis-related genes in OS. CXCL12 might participate in OS progression via leukocyte migration function. BPA might be an important metabolite interacting with multiple prognosis-related genes.

Additional file 3: Table S1. Survival data and results for survival analysis of all feature genes.

Homeobox D 10 (HOXD10) is important in cell differentiation and morphogenesis and serves as a tumor suppressor gene (TSG) in a number of malignancies. The present study investigated its promoter methylation status and association with the clinicopathological features of endometrial cancer (EC), and measured HOXD10 protein expression levels. EC samples (n=62), including 50 endometroid adenocarcinoma (EA) and 12 mucinous endometrial carcinoma samples (EC) and 70 non-cancerous samples were collected. All samples were evaluated for the methylation status of several TSGs, including HOXD10, using methylation-specific PCR. HOXD10 expression level was evaluated using immunohistochemistry. 5-Aza-2-deoxycytidine treatment was performed in the EC cell line Ishikawa to observe the change in HOXD10 expression levels. HOXD10 promoter methylation was more frequent in cancer samples (P<0.001). Downregulation of HOXD10 in EC samples was confirmed at the protein level using immunohistochemistry (P<0.001) and immunohistochemical staining was negatively associated with methylation status (P<0.05). Less HOXD10 protein was expressed in MEC compared with EA samples (P<0.001). The HOXD10 promoter was hypermethylated in both EA and MEC, causing decreased HOXD10 protein expression levels in EC cells. HOXD10 expression levels were partially reversed by 5-Aza-2-deoxycytidine treatment. The results of the present study demonstrated that epigenetic silencing of HOXD10 putatively contributed to the tumorigenesis of EA. Although there was no significant difference in HOXD10 methylation between EA and MEC, HOXD10 protein expression levels differed between these two diseases, indicating that it may be a useful protein biomarker for distinguishing between these two lesions.

BACKGROUND Circular RNAs (circRNAs) are novel non-coding RNAs that have important roles in tumor progression. This study aimed to measure the levels of hsa_circ_0000885 in serum samples and tumor tissue from patients with osteosarcoma compared with controls and to evaluate the findings with disease-free survival and overall survival. MATERIAL AND METHODS Fifty pairs of osteosarcoma tissues and matched adjacent normal tissue were obtained from patients who underwent the same chemotherapy regimen before surgery. Blood samples were obtained from 30 patients with osteosarcoma before and after chemotherapy, 25 patients with osteosarcoma before and after surgery, 27 patients with benign bone tumors, and 25 age-matched and sex-matched healthy controls. Circular RNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) were used to analyze hsa_circ_0000885 expression. RESULTS Hsa_circ_0000885 expression was significantly increased in tissue and serum samples from patients with osteosarcoma, compared with controls, with significantly increased expression levels in patients with Enneking stage IIB and III osteosarcoma, compared with early-stage osteosarcoma. Patients with high serum and tumor levels of hsa_circ_0000885 had lower rates of disease-free survival and overall survival. The serum expression levels of hsa_circ_0000885 were significantly higher in patients with osteosarcoma compared with patients with benign bone tumors or healthy controls. CONCLUSIONS Hsa_circ_0000885 was upregulated in osteosarcoma, and it could serve as a good prognostic biomarker indicating poor clinical outcomes of osteosarcoma. Hsa_circ_0000885 was upregulated in serum of osteosarcoma patients and could serve as a good diagnostic biomarker for osteosarcoma.

Breast cancer is the most common female malignancy worldwide, however its molecular pathogenesis still needs in-depth investigation. Here we first revealed that the olfactory receptor family 2, subfamily T, member 6 (OR2T6) was significantly over-expressed in breast cancer tissues compared with normal breast tissues. OR2T6 expression was tightly correlated with higher TNM staging, positive lymph node metastasis, and associated with poorer patients' overall and disease-free survival. And OR2T6 enhanced the proliferation, invasion, and migration ability of breast cancer cell lines in vitro (MCF-7 and MDA-MD-231). Mechanically, it promoted the expression of mesenchymal markers (Vimentin, N-cadherin, and β-catenin) while inhibited E-cadherin expression, suggesting that OR2T6 played a key role in the regulation of epithelial-mesenchymal transition (EMT) process. Moreover, the human gene expression microarray clarified that MAPK/ERK pathway could be initiated by OR2T6 at mRNA level, which was further confirmed at protein level by western blot analysis. Thus, we concluded that OR2T6, as a novel oncogene, contributed to the progression of breast carcinoma by the initiation of EMT and MAPK/ERK pathway.

Cyclin-dependent kinase subunit (CKS) 2 is a member of the CKS family, which plays an important role in the regulation of meiosis and mitosis. Overexpression of CKS2 has been reported in several types of tumors. However, few studies have investigated its role in uterine leiomyosarcoma (ULMS). In the present study, the expression of CKS2 in 38 cases of ULMS and 38 cases of uterine leiomyoma (ULM) was analyzed by immunohistochemistry. Moreover, the functional analysis of CKS2 was performed in ULMS cell lines. A significantly higher expression of CKS2 was found in ULMS tissues than in ULM tissues (P<0.01) and high CKS2 expression was associated with increased tumor size, low progesterone receptor expression and poor prognosis in patients with ULMS. Multivariate Cox regression analysis revealed that CKS2 expression status was an independent predictor of overall survival for ULMS. Furthermore, silencing of CKS2 in ULMS cells inhibited cell proliferation, colony formation, migration and invasion, and resulted in cell cycle arrest. In conclusion, the present study demonstrated that CKS2 may serve as a marker for the differential diagnosis of ULMS and ULM. In addition, it may act as an independent prognostic factor in patients with ULMS, and serve as a novel target for ULMS therapy.

SAHA, a member of histone deacetylase inhibitors (HDACIs), which emerged as a class of novel antitumor drug, has been used in clinical treatment of cancers. However, clinical experience of SAHA in solid tumors has been disappointing. Nevertheless, the underlying mechanism of this deficiency is not clearly understood. In the present study, we found that SAHA could induce epithelial-mesenchymal transitions (EMT) in lung cancer A549 cells, which was associated with increased migration capability and cellular morphology changes. We showed that SAHA decreased epithelial marker E-cadherin's expression and increased the expression of mesenchymal marker vimentin. SAHA upregulated the protein and mRNA expression of transcription factor Slug in a time-dependent manner and promoted its nuclear translocation. We further demonstrated that SAHA upregulated Slug expression by promoting Slug acetylation but not influencing the phosphorylation of GSK-3β, a main kinase-controlled Slug expression. Finally, silencing of Slug by siRNA reversed EMT marker expressions and cellular morphology change induced by SAHA, suggesting that Slug plays a crucial role in SAHA-mediated EMT in A549 cells. Our research study provided a better understanding of treatment failure of SAHA in patients with solid tumors. Therefore, more attention should be paid to cancer treatment using SAHA and strategies for reversing EMT before using SAHA would be better if the value of SAHA in the treatment of solid tumors, especially lung cancer, is realized.

Background: This study aims to determine the effects of artesunate on proliferation, apoptosis and β-catenin expression in the human osteosarcoma cell line MG-63. Methods: MG-63 cells in the logarithmic growth phase were collected and cultured with different concentrations of artesunate (12.5 µg/mL, 25 µg/mL and 50 µg/mL) for 24 h, 48 h and 72 h. The total number of MG-63 cells and the morphological changes were observed under an inverted microscope. The MTT assay was adopted to test the inhibition rate (IR) of cell growth. The apoptosis rate was detected using annexin V/propidium iodide (PI) staining. Cell cycle distribution was identified by flow cytometry (FCM), and the expression levels of β-catenin, cyclins and cyclin dependent kinases (CDKs) were measured using Western blotting. Results: The results of the MTT assay indicated that artesunate could remarkably inhibit MG-63 cell proliferation compared with the rates in the untreated control group (0 µg/mL artesunate), and the inhibitory effect was dose-dependent. The apoptosis rate of MG-63 cells was elevated as the concentration of artesunate increased, and all the rates were significantly higher than that in the control group. Additionally, as the artesunate concentration increased, the proportion of MG-63 cells in G0/G1 phase gradually declined whereas that of cells in the G2/M and S phases increased. Western blotting confirmed that a higher concentration of artesunate reduced the expression levels of β-catenin, cyclin A, cyclin D1 and CDK1 and increased the expression levels of cyclin B1; however, artesunate had no impact on CDK2 expression in MG-63 cells. Conclusion: These results demonstrated that artesunate can inhibit β-catenin expression and cell proliferation as well as promote cell apoptosis in MG-63 cells, which indicates that artesunate may serve as a promising drug in the clinical treatment of osteosarcoma.