While our prior study identified the HLA-A *0201-restricted ACRBP epitope peptide and demonstrated its capacity to generate cytotoxic T lymphocytes (CTLs) in vitro, the clinical relevance of the peptide-induced T cell reactivity in ovarian cancer (OC) patients and the in vivo anti-tumor efficacy of these CTLs remain unexplored. In this study, dendritic cells were sensitized with ACRBP peptide (ALLVLCYSI) and co-cultured with autologous CD8+T cells to induce the production of specific cytotoxic T lymphocytes (Pep-CTLs). The anti-tumor effects of Pep-CTLs were evaluated in SCID mice bearing human ovarian cancer (OC) OVCAR-3 cells. Concurrently, we co-cultured ALLVLCYSI peptide with peripheral blood mononuclear cells (PBMCs) from OC patients (HLA-A2+, ACRBP+) and assessed the number of specific T cells using ELISPOT assays. The immunological impact of the ACRBP peptide against human OC was validated through both in vitro and in vivo experiments. These findings establish a preclinical foundationfor developing ACRBP peptide-based vaccines in OC immunotherapy. To further elucidate ACRBP’s role in OC treatment, the study analyzed single-cell RNA sequencing data from 8 OC patients and bulk RNA sequencing data from the Cancer Genome Atlas Project (TCGA) comprising 308 ovarian cancer cases. This analysis aimed to explore the heterogeneity among ACRBP-expressing tumor cell populations and to investigate the correlation between ACRBP expression and immune molecule expression (including MHC and chemokines) alongside chemotherapy response. These insights furnish a theoretical framework supporting the future application of ACRBP in tumor immunotherapy and strategies to prevent immune escape.

Abstract Background Tumor immunotherapy has become an important adjuvant therapy after surgery, radiotherapy, and chemotherapy. In recent years, the role of tumor‐associated antigen (TAA) in tumor immunotherapy has become increasingly prominent. Cancer‐testis antigen (CTA) is a kind of TAA that is highly restricted in a variety of tumors and can induce an immune response. Aims This review article aimed to evaluate the role of CTA on the progression of ovarian cancer, its diagnostic efficacy, and the potential for immunotherapy. Methods We analyzed publications and outlined a comprehensive of overview the regulatory mechanism, immunogenicity, clinical expression significance, tumorigenesis, and application prospects of CTA in ovarian cancer, with a particular focus on recent progress in CTA‐based immunotherapy. Results The expression of CTA affects the occurrence, development, and prognosis of ovarian cancer and is closely related to tumor immunity. Conclusion CTA can be used as a biomarker for the diagnosis and prognosis evaluation of ovarian cancer and is an ideal target for antitumor immunotherapy. These findings provide novel insights on CTA in the improvement of diagnosis and treatment for ovarian cancer. The successes, current challenges and future prospects were also discussed to portray its significant potential.

Objective: To screen and validate cancer testis antigens (CTAs) as potential biomarkers and explore their molecular mechanisms in glioblastoma (GBM). Methods: Ribonucleic acid sequencing (RNA-seq) and bioinformatics analyses were utilized to screen the highly expressed CTAs in GBM. Correlation analysis was used to identify poten-tial biomarkers associated with tumor purity and prognosis. Immunohistochemistry was applied for detection of pro-tein expression. Protein-protein interaction (PPI) network construction, functional enrichment analysis, and binding domain prediction were performed to investigate the underlying molecular mechanisms of GBM. Results: A total of 8 highly expressed CTAs were identified in GBM. One of them was PDZ-binding kinase (PBK). PBK messenger RNA (mRNA) was most highly expressed in GBM and associated with tumor purity and prognosis, PBK protein expression was also significantly increased in GBM tissues and correlated with p53 expression. Functional enrichment analysis revealed that the PBK related genes were predominantly enriched in cell cycle pathway with 38 genes enriched. The proteins encoding by these 38 genes were performed by binding domain prediction analysis, which demonstrated 15 proteins interacting with PBK. Most of these proteins were up regulated in GBM. Conclusion: PBK is highly ex-pressed in GBM. It may serve as a potential biomarker for GBM targeting therapy and the cell cycle modulator by interacting with certain key molecules of cell cycle in GBM.

Objective Glioblastoma (GBM) is the most common, invasive, and malignant primary brain tumor with a poor prognosis and high recurrence rate. It’s known that some microRNAs (miRNAs) which are associated with tumorigenesis and progression can be considered as prognostic and therapeutic targets in tumors including GBM. This study aims to highlight the potential role of the core miRNAs in GBM and their potential use as a prognostic and therapeutic biomarker. Methods Differentially expressed miRNAs (DEmiRNAs) were identified in GBM by integrating miRNA-sequencing results and a GBM microarray dataset from the Gene Expression Omnibus (GEO) database through bioinformatics tools. The dysregulated miRNAs were identified by survival analysis through Chinese Glioma Genome Atlas (CGGA). Target genes of the dysregulated miRNAs were predicted on MiRWalk and miRTarBase database. TAM2.0 database, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis were used to analyze the function of the dysregulated miRNAs. Subsequently, protein-protein interaction (PPI) network analysis was used to identify the top 20 hub targets of the up-regulated and down-regulated miRNAs, respectively. Then, core miRNAs in GBM were identified by constructing dysregulated miRNA-differentially expressed hub gene networks. Validation of the core miRNAs expression was detected in 41 GBM tissues compared to 8 normal brain tissues. Furthermore, the potential biomarkers were identified by clinical correlation analysis and survival analysis. Results Totally, 68 intersecting DEmiRNAs were identified, 40 of which were upregulated and the other 28 miRNAs were downregulated. Two upregulated and 4 downregulated miRNAs showed prognostic significance. Most differentially expressed hub genes were regulated by the miR-28-5p and miR-1224-5p, which were respectively upregulated and downregulated in GBM. The correlation between miR-1224-5p level and recurrence was statistically significant ( P =0.011). Survival analysis showed that high miR-28-5p level and high miR-1224-5p level were both associated with better prognosis. Moreover, high miR-1224-5p level was an independent prognosis factor for GBM patients according to the cox regression analysis. Conclusion MiRNA-1224-5p could be a potential target for the prognosis and treatment in GBM.

OBJECTIVE:Glioblastoma multiforme (GBM), the most malignant intracranial neoplasm, is associated with a high mortality and recurrence rate due to the aggressive nature and heterogeneity of the tumor. Some of the molecular markers involved in the tumorigenesis of GBM are essential in prognosis, diagnosis, and treatment. Due to the limitations of therapeutic effects, this study aims to explore novel biomarkers with prognostic value and to provide new insights into therapeutic targets.METHODS:The expression profile of mRNAs in GBM was detected by RNA-sequencing, and differentially expressed genes were identified by integrating the data from RNA-seq results and the GEPIA2 database. Of the total 40 hub genes, FN1, P4HB, and PPIB showed prognostic significance based on both GEPIA2 and CGGA databases. The validation of FN1, P4HB, and PPIB expression by qPCR and correlation analysis with clinicopathological features were performed in 41 GBM tissues from our institution.RESULTS:Kaplan-Meier analysis revealed that FN1 and P4HB expressions levels were related to the overall survival (OS) of GBM patients (P<0.05). Multivariate analysis showed that FN1 overexpression (HR=9.199, P=0.002) was an independent and unfavorable prognostic factor for GBM patients. The median survival time was 8.5 months and 21 months for high and low expressions of FN1, respectively.CONCLUSION:It was suggested that FN1 could be an ideal target for prognosis and a potential therapeutic target in GBM.

Abstract Purpose Ovarian cancer (OC) remains the most lethal gynecological malignancy worldwide. The recrudescence and chemoresistance are main reasons for the high mortality of OC. Identifying effective therapeutic targets is an urgent need. Cancer testis antigens (CTAs) are attractive targets for cancer therapy because of their expression restriction. Methods Based on the RNAseq data of OC in The Cancer Genome Atlas (TCGA) database, we screened the differentially expressed genes of OC from the Cancer-Testis Database and created a protein–protein interaction (PPI) network to analyze co-expressed genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to explore the potential functions of genes. The 3D structure of the protein was simulated and domain interactions were analyzed. We evaluated the expression of candidate gene in OC via qRT-PCR and immunohistochemistry, its correlation with clinical indicators was also analyzed. Results Seven significant CTAs were found to be differentially expressed in OC, PBK was eventually selected as a prognostic factor. The established PPI network contained 93 co-expression genes with 966 protein interactions. Functional enrichment analysis showed that PBK was involved in regulating cell cycle, cell division and mitosis. PBK shares common domains with CDK1, CDK2, PLK1, CCNA2 and CCNB1. PBK was apparently up-regulated in OC, and its overexpression was correlated with the FIGO stage, lymph-node metastasis, and chemoresistance. Conclusion PBK was highly expressed in OC and related to poor prognosis, it functions as a crucial cell-cycle regulator. PBK could be a potential biomarker for OC therapies and prognosis prediction.

Objective The study evaluated the efficacy of combined epigenetic drugs of decitabine (DAC), valproic acid (VPA), and trichostatin A (TSA) on immunotherapy against glioma. Methods The expression and prognosis of MAGE-D4 in glioma were analyzed online, and the expression of MAGE-D4 and HLA-A2 in glioma induced by epigenetic drugs was detected by qRT-PCR, Western blot, and flow cytometry. The methylation status of the MAGE-D4 promoter was determined by pyrosequencing. An HLA-A2 restricted MAGE-D4 peptide was predicted and synthesized. An affinity assay and a peptide/HLA complex stability assay were performed to determine the affinity between peptide and HLA. CCK8 assay, CFSE assay, ELISA and ELISPOT were performed to detect the function of MAGE-D4 peptide-specific T cells. Flow cytometry, ELISA, and cytotoxicity assays were used to detect the cytotoxicity effect of MAGE-D4 peptide-specific T cells combined with epigenetic drugs against glioma in vitro. Finally, the glioma-loaded mouse model was applied to test the inhibitory effect of specific T cells on gliomas in vivo. Results MAGE-D4 was highly expressed in glioma and correlated with poor prognosis. Glioma cells could be induced to express MAGE-D4 and HLA-A2 by epigenetic drugs. MAGE-D4-associated peptides were found that induce DCs to stimulate the highest T-cell activities of proliferation, IL-2 excretion, and IFN-γ secretion. MAGE-D4 peptide-specific T cells treated with TSA only or combining TSA and DAC had the most cytotoxicity effect, and its cytotoxicity effect on glioma cells decreased significantly after HLA blocking. In vivo experiments also confirmed that MAGE-D4-specific T cells inhibit TSA-treated glioma. Conclusion MAGE-D4 is highly expressed in glioma and correlated with the prognosis of glioma. The novel MAGE-D4 peptide identified was capable of inducing MAGE-D4-specific T cells that can effectively inhibit glioma growth, and the epigenetic drug application can enhance this inhibition.

MicroRNA (miRNA), a non-coding single-stranded RNA molecule with 20-23 nucleotides encoded by endogenous genes, plays an essential role in maintaining normal cell function and regulating cell proliferation, differentiation, apoptosis, autophagy, and cell metabolism. The imbalance between miRNA and genes can cause a series of diseases, including malignancies. miRNA-326 (miR-326) is extensively known for its core regulation of various biological processes. This review presents an overview of the highlights of miR-326 in female-related diseases. To understand the impact of miR-326 on female disorders, we search all published studies about miR-326 having a high incidence in female conditions, including cervical cancer, endometrial cancer, breast cancer, intrauterine adhesion, and multiple autoimmune diseases. We aim to learn about the mutual regulation mechanism between miR-326 and related genes and signaling pathways, as well as to elaborate on the value of miR-326 as a potential biomarker and therapeutic target of female diseases. Our results provide reliable evidence and new strategies for treating female tumors and autoimmune diseases.

Abstract Introduction Cancer testis (CT) antigens are attractive targets for cancer immunotherapy because of their expression restriction and immunogenicity. The acrosin binding protein (ACRBP) is a member of CT antigens. This study aimed to evaluate ACRBP expression and immunogenicity in ovarian cancer (OC). Methods The expression level of ACRBP in OC tissues, normal ovarian tissues, and cell lines was detected via quantitative real‐time polymerase chain reaction (qRT‐PCR) and immunohistochemistry. We determined the levels of ACRBP antigen and antibody in serum samples collected from patients with OC and healthy donors using enzyme‐linked immunosorbent assays (ELISA), the level of ACRBP in cell‐cultured medium was also tested. Results ACRBP mRNA and protein expressions were upregulated in OC tissues relative to normal tissue, especially highly expressed in epithelial ovarian cancer (EOC). Moreover, ACRBP expression was significantly correlated with International Federation of Gynecology and Obstetrics (FIGO) stage and chemosensitivity. Serological analysis showed that anti‐ACRBP antibody was detected in the sera of 16 of the 56 (28.5%) patients with OC but not in healthy donors. The area under the receiver operating characteristic curve for ACRBP antibody was 0.802 (95% confidence interval [CI]: 0.708–0.876), and the sensitivity and specificity for ACRBP antibody was 85.71% and 55.0%, respectively. Kaplan–Meier analysis revealed that the overall survival (OS) and disease‐free survival (DFS) in OC patients with high ACRBP expression were significantly lower than those with low expression (p = 0.040, p = 0.021). However, ACRBP antibody level was not associated with prognosis. Conclusion ACRBP expression was upregulated in OC tissues and induced humoral immune response in patients with OC, suggesting that ACRBP is a potential prognostic biomarker and a target of tumor immunotherapy for OC.

Accumulating evidence shows that tumor immune microenvironment infiltration plays a crucial role in the occurrence and development of endometrial carcinoma (EC). At present, studying the regulation mechanism of the immune microenvironment in EC remains a challenge. We calculated the amount of immune and stromal components and the proportion of tumor-infiltrating immune lymphocytes via ESTIMATE, single-sample gene enrichment analysis (ssGSEA), and CIBERSORT computational methods. Differentially expressed genes were analyzed by protein–protein interaction network construction and univariate survival analysis. CD3D expression was negatively correlated with clinicopathological features, such as age, histological type, clinical grade, and pathological stage, but positively correlated with the survival of patients with EC. Logistic regression analysis suggested that CD3D expression could be used as a biomarker to predict EC occurrence. Gene set enrichment analysis revealed that CD3D was upregulated in the immune effect pathway. Interestingly, CD3D was downregulated in the EC pathway. EC was divided into three immune cell subsets by ssGSEA. Correlation analysis between ssGSEA and CIBERSORT showed that the infiltration level of CD8 + T cells and plasma cells in tumor-infiltrating lymphocytes was lower than that in the high-immune-score group (Immunity_H). Moreover, CD3D expression in Immunity_H was low, and the prognosis was even worse. This study demonstrated that low CD3D expression is an index of poor prognosis in patients with EC. Levels of CD3D in tumor microenvironment (TME) might be a useful indicator for predicting the prognosis of patients with EC patients.

目的 探讨肿瘤相关抗原MAGE-D4在肝细胞癌(HCC)中的表达情况,并分析其临床意义.方法 基于癌症基因组图谱(TCGA)数据库,获取371例HCC组织和50例正常肝组织的转录数据.应用数据挖掘工具UALCAN分析HCC组织中MAGE-D4 mRNA的表达情况,并筛选与其表达相关的基因.应用Kaplan-Meier Plotter分析MAGE-D4 mRNA表达水平与患者生存预后的关系.另收集于广西医科大学第一附属医院2009年6月至2011年8月经手术获取的HCC标本70例,癌旁组织标本30例,采用免疫组织化学法(IHC)检测MAGE-D4蛋白的表达情况,并分析其与患者临床指标的关联性.结果 基于TCGA数据库数据的分析结果显示,MAGE-D4 mRNA在HCC组织中的表达水平显著高于正常肝组织(P<0.05),且与甲胎蛋白(AFP)mRNA表达呈正相关(r=0.556,P=0.000).生存分析结果显示,MAGE-D4 mRNA高表达与黄种人HCC患者不良生存预后具有显著关联(HR=1.860,P=0.037).基于临床获取标本的分析结果显示,MAGE-D4蛋白主要定位于细胞质和细胞核,HCC组织的MAGE-D4高表达率显著大于癌旁组织(48.57％vs 23.33％;χ2=5.530,P=0.019).MAGE-D4高表达组HCC患者血清AFP浓度≥400μg/L的人数比例显著大于低表达组(P<0.05),但两组在性别、年龄、肿瘤级别、肿瘤分期、肿瘤直径、癌栓和乙型肝炎病毒(HBV)感染等方面比较差异无统计学意义(P>0.05).结论 MAGE-D4在HCC组织中高表达,其可能在HCC的发生和发展过程中发挥重要作用,有望成为HCC的潜在治疗靶点和预后标志物.

BACKGROUND The family of MAGE genes is well known due to the majority of MAGE genes expressing specifically in tumor tissues while restrictedly in normal tissues. MAGE-D4 is one of the MAGE family and considered as a promising target for glioma immunotherapy because of its overexpression in glioma and restricted expression in normal tissues. Whereas the mechanism of MAGE-D4 heterogeneous expression in glioma has not yet been elucidated. In this study, the transcriptional regulation mechanism of MAGE-D4 in glioma is focused from the perspectives of promoter methylation and SP1. METHODS Dual-luciferase reporter assay was performed to identify the core promoter of MAGE-D4 gene. Mass spectrometry was applied to quantify the methylation status of MAGE-D4 promoter in 50 glioma and 9 normal brain tissues. The influence of methylation and SP1 on MAGE-D4 transcriptional activity was evaluated by dual-luciferase reporter assay, qRT-PCR, western blot and ChIP-qPCR. Decitabine, an epigenetic drug, was used to treat the glioma cells. Then the treated cells were evaluated the influence of demethylation on SP1 binding to MAGE-D4 promoter. RESULTS The -358 to +172 bp region was identified as the core promoter of MAGE-D4 gene which demonstrated hypomethylated and negative correlation between methylation level and MAGE-D4 mRNA expression in glioma tissues. For single CpG unit analysis, 8 CpG units (CpG unit 1, 2, 3, 4, 5, 6, 9 and 12) in MAGE-D4 core promoter showed hypomethylated in glioma and the methylation level of CpG unit 6 was positively associated with the prognosis of glioma patients. Furthermore, the methylation level of CpG unit 1 and 6 was negative negatively correlated with MAGE-D4 mRNA expression. Then, the results demonstrated that the promoter activity of MAGE-D4 was decreased by methylation in glioma cell lines. In addition, SP1 can binds directly to the MAGE-D4 promoter leading to up-regulation of MAGE-D4 mRNA through activation of its promoter. Finally, demethylation of MAGE-D4 promoter could benefit the SP1 binding and resulting co-activation of MAGE-D4 promoter by demethylation and SP1 in glioma cell lines. CONCLUSION These findings indicate that the synergies of promoter hypomethylation and SP1 up-regulated MAGE-D4 transcription in glioma, which implies a potential approach to resolve the heterogeneous expression of MAGE-D4 in order to establish foundation for the MAGE-D4 based glioma therapy.