Journal of Immunology Research

Increased FOXM1 Expression was Associated with the Prognosis and the Recruitment of Neutrophils in Endometrial Cancer

Background. Although the biological functions of Forkhead box protein M1 (FOXM1) were explored in a variety of cancer, to date, however, little attention has been paid to the situation of FOXM1 in EC endometrial cancer (EC). Method. Bioinformatics analysis, including GEPIA, TIMER, cBioPortal, LinkedOmics, and STRING were used to analyze the FOXM1 gene expression, genetic alteration, and immune cell infiltration in EC. IHC staining, qPCR, cell viability, and migration assay were applied to identify the functions of FOXM1 in EC. Results. FOXM1 was highly expressed in EC tissues and closely correlated with the prognosis of EC patients. FOXM1 knockdown inhibited EC cell proliferation and invasion as well as migration. FOXM1 genetic alteration was verified in EC patients. Coexpression network of FOXM1 indicated that it had roles in the EC cell cycle and the infiltration of immune cells in EC. Furthermore, bioinformatic and immunohistochemical analysis indicated that FOXM1 induced the increased CD276 expression and also enhanced the neutrophil recruitment in EC. Conclusion. Our present study discovered a novel role of FOXM1 in EC, suggesting FOXM1 could be treated as a potential prognostic biomarker and immunotherapeutic target in EC diagnosis and treatment.

RIPK4 Is an Immune Regulating-Associated Biomarker for Ovarian Cancer and Possesses Generalization Value in Pan-Cancer

Ovarian cancer (OC) is the most lethal gynecologic cancer. Many studies have reported that RIPK4 (receptor interacting serine/threonine kinase 4) displayed a dysregulated level in many types of tumors. However, its expressions and functions in OC were rarely reported. The levels of RIPK4 were detected in OC and nontumor specimens using TCGA and GEO datasets. The prognostic values of RIPK4 in patients were determined using Kaplan-Meier methods and Kaplan-Meier assays. GO assays and KEGG pathway assays were carried out for functional enrichments. CIBERSORT was applied for estimating the fractions of immune cell types. Finally, RIPK4 was validated in pan-cancer. In this study, our group found that RIPK4 exhibited a higher level of RIPK4 in OC specimens than nontumor specimens. Survival studies revealed that patients with high RIPK4 expressions showed a shorter overall survival than those with low RIPK4 expression. Multivariate assays further confirmed that RIPK4 expression was an independent prognostic element for OC. KEGG pathway analysis displayed that the dysregulated genes in specimens with high RIPK4 expressions were enriched in focal adhesion, proteoglycans in cancer, central carbon metabolism in cancer, and insulin secretion. Correlation analyses showed that several TICs were positively correlated with RIPK4 expression. The pan-cancer validation results showed that RIPK4 was associated with survival in five tumors. Overall, our findings suggested RIPK4 as a prognostic marker in OC.

lncRNA OIP5-AS1 Suppresses Cell Proliferation and Invasion of Endometrial Cancer by Regulating PTEN/AKT via Sponging miR-200c-3p

Background. Endometrial carcinoma (EC) is one of the major gynecologic malignancy cancers affecting females with dismal prognosis and high mortality around the world. Numerous studies have proven that an aberrant level of long noncoding RNAs is present in many endometrial cancer patients, while the underlying molecular mechanism remains unclear. Method. The expression levels of lncRNA OIP5-AS1, miR200c-3p, and PTEN were measured by a quantitative real-time polymerase chain reaction in endometrial cancer tissue and endometrial cancer cells. CCK8 assay, wound-healing assay, and cell colony formation were applied to evaluate cell proliferation, cell migration, and cell colony formation ability. Cell cycle and cell apoptosis were detected by flow cytometry. The interactions between OIP5-AS1, miR200c-3p, and PTEN were explored by luciferase activity. Results. In the present study, we demonstrated that long noncoding RNA OIP5-AS1 was significantly reduced in EC tissue compared with normal tissue. The lower expression level of OIP5-AS1 was also confirmed in four kinds of EC cell lines compared with the normal endometrial cell line. Gain- and loss-of-function of experiments indicated that upregulation of OIP5-AS1 could inhibit the proliferation, migration, and invasion of EC cells in vitro. Meanwhile, overexpression of OIP5-AS1 could also suppress the growth of tumor in the xenograft model. Moreover, further study revealed that miR-200c-3p could bind to OIP5-AS1, and the loss function of miR-200c-3p could reverse the elevated OIP5-AS1’s inhibitory effect on the progression of EC. Furthermore, we found that downregulation of miR-200c-3p was inversely correlated with PTEN expression in EC cells. Reduced OIP5-AS1 could lead to the accumulation of miR-200c-3p, which could induce the upregulation of PTEN indirectly. Conclusion. Our study demonstrated a novel molecular mechanism that lncRNA OIP5-AS1 could modulate the progression of EC by combining competitively with miR-200c-3p to control the PTEN/AKT pathway in EC cells, which might supply important information for developing novel therapeutic strategies for EC patients.

KIF26B and CREB3L1 Derived from Immunoscore Could Inhibit the Progression of Ovarian Cancer

Background . Ovarian cancer (OV) is characteristic of high incidence rate and fatality rate in the malignant tumors of female reproductive system. Researches on pathogenesis and therapeutic targets for OV need to be continued. This study mainly analyzed the immune‐related pathogenesis and discovered the key immunotherapy targets for OV. Methods . WGCNA was used for excavating hub gene modules and hub genes related to the immunity of OV. Enrichment analysis was aimed to analyze the related pathways of hub gene modules. Biological experiments were used for exploring the effect of hub genes on SKOV3 cells. Results . We identified two hub gene modules related to the immunoscore of OV and found that these genes in the modules were related to the extracellular matrix and viral infections. At the same time, we also discovered six hub genes related to the immunity of OV. Among them, KIF26B and CREB3L1 can affect the proliferation, migration, and invasion of SKOV3 cells by the Wnt/ β ‐catenin pathway. Conclusions . The local infection or inflammation of ovarian may affect the immunity of OV. KIF26B and CREB3L1 are expected to be potential targets for the immunotherapy of OV.

FBXL16 Promotes Endometrial Progesterone Resistance via PP2AB55α/Cyclin D1 Axis in Ishikawa

Background. F-box proteins are essential components of the E3 ubiquitin ligases which are involved in the regulation of almost all life activities such as cell cycle, proliferation, and apoptosis, which have become an important research and drug target. However, there are few studies on F-box and leucine-rich repeat protein 16 (FBXL16) in endometrial carcinoma. Methods. Clinical samples were collected for determining the correlation between FBXL16 and endometrial carcinoma. Cells were screened and established with Ishikawa cells which proved the fundamental role of FBXL16 in regulating cell proliferation and cell cycle. The MPA-resistant endometrial carcinoma cell line Ishikawa/MPA was established. FBXL16, PP2AB55α, and cyclin D1 were analyzed separately in MPA sensitive and resistant Ishikawa cells in vitro and in vivo. Results. The high expression of FBXL16 was positively correlated with MPA resistance and poor prognosis of endometrial cancer. MPA tolerance of endometrial cancer cells was inhibited by knockdown of FBXL16 in DNA content assessment, CCK-8, and colony formation. It was confirmed that FBXL16 inhibited the activity of substrate PP2AB55α by binding to PP2A, reduced the phosphorylation level at Thr308 site of AKT1, inhibited the expression of GSK-3β, and thus led to a significant decrease in the phosphorylation level of cyclin D1, which prevented the ubiquitination recognition and degradation of cyclin D1. Conclusion. In our experiments, FBXL16 binds PP2A to promote the dephosphorylation of Thr286 site of cyclin D1 via AKT1/GSK3β/cyclin D1 pathway, which is required for resisting the ubiquitination degradation and enhances the MPA resistance of Ishikawa.

BCHE as a Prognostic Biomarker in Endometrial Cancer and Its Correlation with Immunity

Background. In developed countries, the most common gynecologic malignancy is endometrial carcinoma (EC), making the identification of EC biomarkers extremely essential. As a natural enzyme, butyrylcholinesterase (BCHE) is found in hepatocytes and plasma. There is a strong correlation between BCHE gene mutations and cancers and other diseases. The aim of this study was to analyze the role of BCHE in patients with EC. Methods. A variety of analyses were conducted on The Cancer Genome Atlas (TCGA) data, including differential expression analysis, enrichment analysis, immunity, clinicopathology, and survival analysis. The Gene Expression Omnibus (GEO) database was used to validate outcomes. Using R tools, Gene Set Enrichment Analysis (GSEA) and Gene Ontology (GO) analyses revealed the potential mechanisms of BCHE in EC. Sangerbox tools were used to delve into the relations between BCHE expression and tumor microenvironment, including microsatellite instability (MSI), tumor neoantigen count (TNC), and tumor mutation burden (TMB). BCHE’s genetic alteration analysis was conducted by cBioPortal. In addition, the Human Protein Atlas (HPA) was used to validate the outcomes by immunohistochemistry, and an analysis of the protein-protein interaction network (PPI) was performed with the help of the STRING database. Results. Based on our results, BCHE was a significant independent prognostic factor for patients with EC. The prognosis with EC was affected by age, stage, grade, histological type, and BCHE. GSEA showed that BCHE was closely related to pathways regulating immune response, including transforming growth factor-β (TGF-β) signaling pathways and cancer immunotherapy through PD1 blockade pathways. The immune analysis revealed that CD4+ regulatory T cells (Tregs) were negatively correlated with BCHE expression and the immune checkpoint molecules CD28, ADORA2A, BTNL2, and TNFRSF18 were all significantly related to BCHE. BCHE expression was also associated with TMB by genetic alteration analysis. Conclusions. Identifying BCHE as a biomarker for EC might help predict its prognosis and could have important implications for immunotherapy.

IL6 Induces mtDNA Leakage to Affect the Immune Escape of Endometrial Carcinoma via cGAS-STING

Endometrial carcinoma (EC) is a commonly diagnosed gynecological malignancy. Interleukin-6 (IL6) plays a critical role in modulating the progression of several types of tumors, including EC. However, the specific mechanism of IL6 in regulating EC progression has not been clearly elucidated. In this study, we performed a series of functional experiments to explore the potential mechanisms involved in IL6 function in the progression of EC. Here, we found that IL6 increased reactive oxygen species (ROS) generation by enhancing the NADPH oxidase (NOX) level and induced mtDNA leakage in EC cells, which further caused the activation of the downstream cGAS-STING signaling and increased production of extracellular vesicle (EV) production from EC cells. Besides, the activation of cGAS-STING signaling enhanced the expression of type I IFN and its downstream molecule PD-L1 through the TBK1-IRF3 pathway. Importantly, a high level mtDNA and PD-L1 were present in EVs derived from IL6-induced EC cells; these vesicles were shown to be able to induce T cell apoptosis. Finally, anti-PD-L1 treatment in mice showed that blockade of PD-L1 significantly reversed tumor immune escape mediated by IL6-induced EVs. Together, we provide evidence that IL6 induced mtDNA leakage to regulate the immune escape of EC cells. Our findings may provide a novel clue for the development of therapeutic targets for EC.

Identification of Five m6A-Related lncRNA Genes as Prognostic Markers for Endometrial Cancer Based on TCGA Database

Background. Due to difficulties involved in its early diagnosis and adequate prognostication, uterine corpus endometrial carcinoma (UCEC) is one of the most serious threats to human health, with the five-year survival rate being as low as roughly 60%. The discovery of specific biomarkers that serve as prognosticators of UCEC is of great significance. The role of N6-methyladenosine- (m6A-) related long noncoding RNAs (lncRNAs) in the pathogenesis of UCEC remains undefined. In this study, we explored the expression profiles of m6A-related lncRNAs of patients with UCEC and identified novel prognostic markers for UCEC. Methods. Gene expression and clinical data were extracted from The Cancer Genome Atlas. Coexpression analysis was performed to identify m6A-related lncRNAs, which were entered into univariate Cox regression models for evaluating the prognosis of UCEC. Clusters of UCEC patients and enrichment pathways were identified using consistent data clustering and gene set enrichment analysis (GSEA). A risk score model was established, and Kaplan–Meier analysis was conducted for investigating overall survival (OS) across two patient groups (high risk and low risk). Lastly, the relationship between the risk score and the cell content of 22 types of immune cells, clusters, age, programmed cell death 1 ligand-1 (PD-L1) expression level, immune score, and pathological grade was analyzed. Results. We identified a total of 2084 lncRNAs associated with m6A, of which 32 lncRNAs were prognostically relevant. Two clusters (clusters 1 and 2) of patients with UCEC were defined; patients in cluster 1 were found to have significantly higher pathological grades and shorter overall survival time compared to those in cluster 2. GSEA showed that “MITOTIC SPINDLE and other pathways” were more enriched in cluster 1. Five major lncRNAs associated with m6A were screened out, and risk score modeling was used for UCEC prognosis prediction. High risk scores were associated with a shorter OS. The risk score was also verified as an independent prognostic indicator for UCEC and was related to immune cell infiltration levels. Finally, we observed a higher pathological grade and greater levels of PD-L1 in the high-risk group than in the low-risk group of patients. Conclusions. m6A-related lncRNAs play an important role in UCEC progression. The risk-based model constructed from the five key m6A-related lncRNAs was implicated in immune cell infiltration and can potentially be an accurate prognosticator for UCEC.

circSLC6A6 Sponges miR-497-5p to Promote Endometrial Cancer Progression via the PI4KB/Hedgehog Axis

Background. As a new kind of noncoding RNAs, circular RNAs (circRNAs) have been substantiated to be involved in multiple biological processes. Accumulating studies indicate that circular RNAs (circRNAs) regulate the development of cancers by acting as miRNA sponges. However, the role of circRNAs in endometrial cancer (EC) is rarely reported. This study was aimed at investigating the functional roles of circSLC6A6 in EC. Methods. The qRT-PCR assay was performed to detect the circSLC6A6 expression in EC tissues and cell lines. The luciferase reporter assay was performed to explore the connection between circSLC6A6 and miR-497-5p as well as the connection between miR-497-5p and PI4KB. The colony formation assay, EdU assay, wound healing assay, and transwell assay were performed to examine the proliferation, migration, and invasion of EC cells. The in vivo assay was performed to reveal the function of circSLC6A6 in tumorigenesis. Results. We found that circSLC6A6 was highly expressed in both EC tissues and cells. And circSLC6A6 promoted the proliferation, migration, and invasion of EC cells in vitro. In vivo, circSLC6A6 promoted tumor growth. Besides, a mechanistic study demonstrated that circSLC6A6 could regulate tumor-associated signaling PI4KB/hedgehog pathway by sponging miR-497-5p. Conclusion. This study illustrates that circSLC6A6 plays a role in promoting EC progression via the miR-497-5p-mediated PI4KB/hedgehog pathway. Our study may provide a potential novel biomarker for EC diagnosis or treatment.

Endometrial Cancer Cells Promote M2‐Like Macrophage Polarization by Delivering Exosomal miRNA‐21 under Hypoxia Condition

Increasing evidence has demonstrated that hypoxia was an aggressive feature in endometrial cancer (EC), which is significantly associated with the tumor grade, lymph node metastasis, and tumor resistance to chemotherapy. However, the relationship between hypoxia and the immune microenvironment in EC is not very clear. Exosomes are small membrane vesicles secreted from a variety of cell types which mediate cell‐to‐cell communication through transported biomolecules. Here, we investigated whether exosomes can play an immunomodulatory role in intercellular communication between EC cells and macrophages. EC KEL cells were cultured under hypoxia or normoxic condition to collect exosomes. After identification, the exosomes derived from hypoxic or normoxic KEL cells were cultured with the monocyte cell line THP‐1 to study the immunoregulation function of KEL cells. The results showed that the total number of exosomes produced by hypoxic KEL cells was significantly higher than that in normoxic condition. In addition, hypoxia markedly stimulated the increase in miRNA‐21 expression in the exosomes. After coculture, we found that exosomal miRNA‐21 could be horizontally transferred into THP‐1 cells. And then, the notably enhanced mRNA expression levels of IL‐10 and CD206 in THP‐1 cells were observed, suggestive of M2 polarization. To further study the effect of miRNA‐21‐containing exosomes, we transfected miRNA‐21 mimics or inhibitor into THP‐1 cells. The results showed that miRNA‐21 mimics promoted IL‐10 and CD206 mRNA expression levels, and the miRNA‐21 inhibitor significantly prevented the alteration induced by intake of hypoxic KEL cell‐derived exosomes. In summary, we found that endometrial cancer KEL cells in hypoxic condition promoted monocyte THP‐1 cell transformation to M2‐like polarization macrophages through delivering exosomal miRNA‐21, which may be a potential mechanism of the formation of the immune microenvironment in EC progression.

Systematic Analysis of Peripheral Immune Signatures and Diagnostic Model Construction in Patients With Uterine Fibroids

Objective To systematically characterize the peripheral immune signature of uterine fibroids (UFs) and to develop a diagnostic model for differentiating UF patients from healthy individuals, thereby providing new insights into UF immunopathogenesis. Methods We performed multiparametric flow cytometry analysis on peripheral blood samples from 31 UF patients and 63 age‐matched healthy controls (HCs). A total of 70 immune parameters were evaluated, encompassing T cells, B cells, natural killer (NK) cells, γδ T cells, and their functional subsets. Results Comprehensive immunophenotyping revealed a distinct peripheral immune profile in UF patients. Key findings included a significant dysregulation within helper T (Th) cell compartments, characterized by elevated frequencies of functional Th and Th17 cells, alongside reduced proportions of senescent Th, T follicular helper 1 (Tfh1), and peripheral Th (Tph) cells. Concurrently, a significant expansion of the B cell compartment was observed, marked by increased total B cells, naïve B cells, immature regulatory B cells (Breg), and transformed B cells. In contrast, the frequencies and functional subsets of cytotoxic T (Tc) cells, γδ T cells, and NK cells showed no significant alterations after false discovery rate (FDR) correction. A random forest (RF) model incorporating key immune markers effectively discriminated UF patients from HCs, identifying several markers as central features with both diagnostic and mechanistic relevance. Conclusion This study presents the first systematic atlas of the peripheral immune landscape in UF, revealing a pattern of systemic immune dysregulation centered on Th and B cell pathways. These findings advance our understanding of the immunopathogenesis of UF and establish a foundation for future immune‐based diagnostic and therapeutic strategies.

Identification of an Immune Gene-Based Cisplatin Response Model and CD27 as a Therapeutic Target against Cisplatin Resistance for Ovarian Cancer

Objective. Evidence demonstrates that the immune microenvironment is extensively associated with chemotherapy response of ovarian cancer (OV). Herein, this study is aimed at establishing a cisplatin response prediction model for OV on the basis of immune genes. Methods. The expression profiles of cisplatin-sensitive and cisplatin-resistant OV specimens were integrated from multiple public datasets. The abundance scores of 22 immune cells were estimated with CIBERSORT algorithm. Differentially expressed immune genes (DEGs) were determined between cisplatin-sensitive and cisplatin-resistant groups. Thereafter, a cisplatin response model was constructed based on prognostic DEGs with logistic regression analysis. The prediction performance was validated in independent cohorts. The possible relationships between the model and immunotherapy were then assessed. Results. Treg scores were significantly decreased in cisplatin-resistant than cisplatin-sensitive OV specimens, with the opposite results for naïve B cells and activated dendritic cells. Fourteen prognostic DEGs were identified and used to develop a cisplatin-response model. The response scores, estimated by the model, showed favorable performance in discriminating cisplatin-response and nonresponse samples. The response scores also presented significantly negative correlations with three well-known cisplatin-resistant pathways and a positive correlation with the expression of CD274 (PD-L1). Moreover, the decreased CD27 expression was observed in cisplatin-resistant groups, and OV specimens with higher CD27 expressions were more sensitive to cisplatin treatment. Conclusion. Altogether, our findings proposed a cisplatin response prediction model and identified CD27 that might be involved in cisplatin resistance. Further investigations suggested that CD27 could be a promising immunotherapeutic target for cisplatin-resistant subset of OV.

Macrophages Phenotype Regulated by IL-6 Are Associated with the Prognosis of Platinum-Resistant Serous Ovarian Cancer: Integrated Analysis of Clinical Trial and Omics

Background. The treatment of platinum-resistant recurrent ovarian cancer (PROC) is a clinical challenge and a hot topic. Tumor microenvironment (TME) as a key factor promoting ovarian cancer progression. Macrophage is a component of TME, and it has been reported that macrophage phenotype is related to the development of PROC. However, the mechanism underlying macrophage polarization and whether macrophage phenotype can be used as a prognostic indicator of PROC remains unclear. Methods. We used ESTIMATE to calculate the number of immune and stromal components in high-grade serous ovarian cancer (HGSOC) cases from The Cancer Genome Atlas database. The differential expression genes (DEGs) were analyzed via protein–protein interaction network, Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) analysis to reveal major pathways of DEGs. CD80 was selected for survival analysis. IL-6 was selected for gene set enrichment analysis (GSEA). A subsequent cohort study was performed to confirm the correlation of IL-6 expression with macrophage phenotype in peripheral blood and to explore the clinical utility of macrophage phenotype for the prognosis of PROC patients. Results. A total of 993 intersecting genes were identified as candidates for further survival analysis. Further analysis revealed that CD80 expression was positively correlated with the survival of HGSOC patients. The results of GO and KEGG analysis suggested that macrophage polarization could be regulated via chemokine pathway and cytokine–cytokine receptor interaction. GSEA showed that the genes were mainly enriched in IL-6-STAT-3. Correlation analysis for the proportion of tumor infiltration macrophages revealed that M2 was correlated with IL-6. The results of a cohort study demonstrated that the regulation of macrophage phenotype by IL-6 is bidirectional. The high M1% was a protective factor for progression-free survival. Conclusion. Thus, the macrophage phenotype is a prognostic indicator in PROC patients, possibly via a hyperactive IL-6-related pathway, providing an additional clue for the therapeutic intervention of PROC.

Exosome-Associated Gene Signature for Predicting the Prognosis of Ovarian Cancer Patients

Background. The exosome is of vital importance throughout the entire progression of cancer. Because of the lack of effective biomarkers in ovarian cancer (OV), we intend to investigate the connection between exosomes and tumor immune microenvironment to verify that exosome-related genes (ERGs) can precisely forecast the prognosis of OV patients. Methods. First, 117 ERGs in The Cancer Genome Atlas (TCGA) dataset were recognized. Afterwards, the risk signature consisting of four ERGs with prognostic significance was built by univariate Cox, least absolute shrinkage and selection operator (LASSO), and multivariate Cox regression analysis. We also validated the risk signature by Kaplan-Meier analysis, receiver operating characteristic curve analysis and principal component analysis. Furthermore, gene set enrichment analysis was performed to compare the enrichment patterns between the two risk subgroups. The connections between the exosome-related gene risk score (ERGRS) and clinical features, immune infiltration, immune checkpoint-related genes, copy number variation, and drug sensitivity were explored. We also assessed the function of the ERGRS to forecast immunotherapeutic efficacy by immunophenoscore (IPS). Results. The high-risk group had a worse prognosis than the group with low risk. We verified that the established model possessed a relatively good prognostic value. Pathway enrichment analysis indicated that the genome-wide group with low risk was enriched in immune-related pathways. We discovered that resting dendritic cells and stromal scores were upregulated in patients with high risk in the TCGA and Gene Expression Omnibus (GEO) cohorts. Moreover, the expression of six common immune checkpoint inhibitor targets was assessed, which revealed that the expression levels of CD274 (PD-L1), PDCD1 (PD-1), and IDO1 in patients with high risk were lower than those in patients with low risk. Afterwards, the low-risk group had higher IPS across the four immunotherapies, implying that it had better effects of immunotherapies. Conclusion. Our study demonstrates that the exosome-related gene risk model is closely associated with immune infiltration. It can well forecast the prognosis of OV patients and guide the selection of immunotherapeutic strategies.

Prognostic and Clinical Value of Interleukin 6 and CD45 + CD14 + Inflammatory Cells with PD‐L1 + /PD‐L2 + Expression in Patients with Different Manifestation of Ovarian Cancer

Ovarian cancer (OC) is one of the deadliest gynecological cancers. Recent studies suggest a crucial role of inflammatory immune system cells in the progression and metastasis of OC. The understanding of inflammatory mechanisms is pivotal for the selection of a biomarker that allows the differentiation between malignant and benign tumors, monitoring the progression of the disease, and identification of patients that will respond to implemented treatment. Our study is aimed at evaluating the profile of IL‐6 in the plasma and peritoneal fluid (PF) of patients with various clinical manifestations of OC ( n = 78). We also examined the relationship between IL‐6 and PD‐L1/PD‐L2 positive CD45 + CD14 + inflammatory cell (MO/MA) levels in three OC environments (TME): peripheral blood (PB), PF, and tumor (TT) and their clinical and prognostic relevance in OC patients. The expression of PD‐L1/PD‐L2 molecules was analyzed by flow cytometry. The IL‐6 levels were determined by ELISA. We found an elevated level of PD‐L1/PD‐L2 positive MO/MA in TT compared to PB ( p < 0.0001). Significantly higher ( p < 0.0001) levels of IL‐6 were observed in PF of the OC patients than in the benign ovarian tumor group ( n = 31). Additionally, we found higher IL‐6 levels in PF than in the plasma of the OC patients. Interestingly, accumulation of IL‐6 was observed in PF of patients with low‐differentiated OC and correlated with worse prognosis. Moreover, we observed correlations between the level of IL‐6 and CD45 + CD14 + cells and between CD45 + CD14 + PD‐L1 + cells and the IL‐6 level in PF. For the first time, we discovered that the higher percentage of CD45 + CD14 + PD‐L2 + cells in PF predicts better survival of OC patients. Our study suggests that CD45 + CD14 + PD‐L2 + cells and IL‐6 may be predictive biomarkers for OC patients. Understanding how the composition of TME changes during OC development and progression is a prerequisite for projecting new therapeutic strategies. Overall, further validation research is warranted.

STK3 Suppresses Ovarian Cancer Progression by Activating NF‐ κ B Signaling to Recruit CD8 + T‐Cells

Serine/threonine protein kinase‐3 (STK3) is a critical molecule of the Hippo pathway but little is known about its biological functions in the ovarian cancer development. We demonstrated the roles of STK3 in ovarian cancer. Existing databases were used to study the expression profile of STK3. STK3 was significantly downregulated in OC patients, and the low STK3 expression was correlated with a poor prognosis. In vitro cell proliferation, apoptosis, and migration assays, and in vivo subcutaneous xenograft tumor models were used to determine the roles of STK3. The overexpression of STK3 significantly inhibited cell proliferation, apoptosis, and migration of ovarian cancer cells in vitro and tumor growth in vivo. Bisulfite sequencing PCR analysis was performed to validate the methylation of STK3 in ovarian cancer. RNA sequencing and gene set enrichment analysis (GSEA) were used to compare the transcriptome changes in the STK3 overexpression ovarian cancer and control cells. The signaling pathway was analyzed by western blotting. STK3 promoted the migration of CD8 + T‐cells by activating nuclear transcription factor κ B (NF‐ κ B) signaling. STK3 is a potential predictor of OC. It plays an important role in suppressing tumor growth of ovarian cancer and in chemotaxis of CD8 + T‐cells.

Profound Functional Suppression of Tumor‐Infiltrating T‐Cells in Ovarian Cancer Patients Can Be Reversed Using PD‐1‐Blocking Antibodies or DARPin® Proteins

PD‐1/PD‐L1 blockade has revolutionized the field of immunooncology. Despite the relative success, the response rate to anti‐PD‐1 therapy requires further improvements. Our aim was to explore the enhancement of T‐cell function by using novel PD‐1‐blocking proteins and compare with clinically approved monoclonal antibodies (mAbs). We isolated T‐cells from the ascites and tumor of 17 patients with advanced epithelial ovarian cancer (EOC) and analyzed the effects using the mAbs nivolumab and pembrolizumab and two novel engineered ankyrin repeat proteins (DARPin® proteins). PD‐1 blockade with either mAb or DARPin® molecule significantly increased the release of IFN‐ γ , granzyme B, IL‐2, and TNF‐ α , demonstrating successful reinvigoration. The monovalent DARPin ® protein was less effective compared to its bivalent equivalent, demonstrating that bivalency brings an additional benefit to PD‐1 blockade. Overall, we found a higher fold increase of lymphokine secretion in response to the PD‐1 blockade by tumor‐derived T‐cells; however, the absolute amounts were significantly lower compared to the release from ascites‐derived T‐cells. Our results demonstrate that PD‐1 blockade can only partially reinvigorate functionally suppressed T‐cells from EOC patients. This warrants further investigation preferably in combination with other therapeutics. The study provides an early pilot proof‐of‐concept for the potential use of DARPin ® proteins as eligible alternative scaffold proteins to block PD‐1.

Comprehensive Analysis of Prognostic Value of MEX3A and Its Relationship with Immune Infiltrates in Ovarian Cancer

MEX3A is a critical RNA-binding ubiquitin ligase that is upregulated in various types of cancer. However, the correlations of MEX3A with prognosis and its molecular mechanism in ovarian cancer (OC) remain unclear. The expression level, prognostic values, and the genetic variations of MEX3A were analyzed via Gene Expression Profiling Interactive Analysis (GEPIA) Oncomine, Kaplan–Meier plotter, and cBioPortal. We used the LinkedOmics database to investigate the functions of MEX3A coexpressed genes and performed visualizing gene interaction network analysis on the GeneMANIA website. The correlations between MEX3A and cancer immune infiltration were analyzed by the Tumor Immune Estimation Resource (TIMER) site and the TISIDB database. Furthermore, in vitro analysis was performed to evaluate the biological functions of MEX3A in OC cells. Our study showed that the expression of the MEX3A in OC was higher than in normal tissues; it had the greatest prognostic value in OC, and strong physical interaction with PABPC1, LAMTOR2, KHDRBS2, and IGF2BP2, which indicated the association between MEX3A and immune infiltration. We also found that MEX3A was negatively related to infiltrating levels of several types of immune cells, including macrophages, neutrophils, dendritic cells (DCs), B cells, and CD8+ T cells. Additionally, in vitro experiments demonstrated that MEX3A promotes proliferation and migration in OC cells. Taken together, MEX3A might influence the biological functions of OC cells by regulating the immune infiltration in the microenvironment as a prognostic biomarker and a potential therapeutic target.

DGAT1 Expression Promotes Ovarian Cancer Progression and Is Associated with Poor Prognosis

Background. Ovarian cancer is the most fatal gynecological malignancy. Owing to its insidious onset, rapid development, and poor prognosis, ovarian cancer is the fifth most common cause of death in women. Although immunotherapy-related drugs, such as Olaparib, can alleviate ovarian cancer progression, there are no remarkable breakthroughs for its effective treatment. It is considered that the transformation of normal cells to cancerous ones involves “recoding” of certain metabolic pathways. Diacylglycerol O-acyltransferase 1 (DGAT1) can synthesize triglycerides by transferring acyl-CoA to diacylglycerol, which plays a key role in lipid synthesis. However, the role of DGAT1 in ovarian cancer is not yet elucidated. Materials and Methods. We analyzed the correlation between DGAT1 and ovarian cancer staging, grading, vascular invasion, and prognosis by collating the information of ovarian cancer specimens from The Cancer Genome Atlas (TCGA) database. Furthermore, the effects of DGAT1 expression on proliferation, migration, invasion, and tumor growth were studied using ovarian cancer cell lines. GSEA was used to analyze the KEGG pathways and biological function enriched because of DGAT1 expression in ovarian cancer. Results. The expression of DGAT1 was elevated in advanced ( p = 0.0432 ), poorly differentiated ( p = 0.0148 ), and vascular invaded ( p = 0.0002 ) ovarian cancer specimens. Prognosis among patients with high expression of DGAT1 was poor. After DGAT1 expression was interfered, proliferation, migration, invasion, colony forming, and tumor growth of ovarian cancer cells were inhibited. In addition, GSEA showed that DGAT1 may be involved in the immune process. Conclusion. DGAT1 expression is associated with the clinical phenotype of ovarian cancer. We suggest that DGAT1 has potential implications in the treatment of ovarian cancer.

Ethanol Extracts of Solanum lyratum Thunb Regulate Ovarian Cancer Cell Proliferation, Apoptosis, and Epithelial-to-Mesenchymal Transition (EMT) via the ROS-Mediated p53 Pathway

Ovarian cancer is a type of common gynecological tumors with high incidence and poor survival. The anticancer effects of the traditional Chinese medicine Solanum lyratum Thunb (SLT) have been intensively investigated in various cancers but in ovarian cancer is rare. The current study is aimed at investigating the effect of SLT on ovarian cancer cells. Lactate dehydrogenase (LDH) and MTT assays indicated that SLT concentrations of 0.25 and 0.5 μg/mL were not cytotoxic and had significant inhibitory effects on the cell viabilities of A2780 and SKOV3 cells, hence were used for subsequent experiments. Flow cytometric and western blot analysis revealed that SLT effectively suppressed ovarian cancer cell proliferation via inducing cell cycle arrest and increasing apoptosis. Cell cycle and apoptosis-related protein expressions were also regulated in SLT-treated cells. Moreover, DCFH-DA and western blot assays demonstrated that SLT enhanced ROS accumulation and subsequently activated the p53 signaling pathway. However, SLT-regulated ovarian cancer cell proliferation, apoptosis, migration, invasion, and EMT were significantly reversed by an ROS inhibitor (NAC, N-acetyl-L-cysteine). Furthermore, A2780 and SKOV3 cells cocultured with M0 macrophages showed that SLT activated the polarization of M0 macrophages to M1 macrophages and inhibited the polarization to M2 macrophages, with the increased percentage of CD86+ cells and decreased percentage of CD206+ cells were detected. In summary, this study illustrated the anticancer effects of SLT on ovarian cancer cells, suggesting that SLT may have the potential to provide basic evidence for the discovery of antiovarian cancer agents.

Downregulation of RIPK4 Expression Inhibits Epithelial-Mesenchymal Transition in Ovarian Cancer through IL-6

RIPK4 has been implicated in multiple cancer types, but its role in ovarian cancer (OC) has not been clearly elucidated. Our data from Gene Expression Profiling Interactive Analysis, RT-PCR, and immunohistochemical analysis showed that RIPK4 was expressed at higher levels in OC tissues and cells than in normal ovarian tissues and cells. Increased RIPK4 expression in OC markedly correlated with a worse overall survival than lower RIPK4 expression levels (hazard rate (HR) 1.5 (1.45–1.87); P = 0.001 ). In functional experiments, RIPK4 downregulation significantly inhibited metastatic behaviours in OC cells. Subsequently, based on data from 593 OC patients in the TCGA database, gene set enrichment analysis revealed that RIPK4 was involved in epithelial-mesenchymal transition (EMT) in OC. At the molecular level, silencing RIPK4 significantly downregulated vimentin, N-cadherin, and Twist expression but induced an increase in the protein level of E-cadherin and inhibited the IL-6 and STAT3 levels. Moreover, IL-6 levels were significantly decreased in RIPK4-silenced OC cells ( P < 0.05 ). The addition of IL-6 to OC cells rescued the suppressive effect of RIPK4 knockdown on EMT. Thus, our data illustrate that downregulation of RIPK4 expression can restrain EMT in OC by inhibiting IL-6. This finding may provide a novel diagnostic and therapeutic target for improving the poor prognoses of OC patients.

Use of Nomogram to Predict the Risk of Lymph Node Metastasis among Patients with Cervical Adenocarcinoma

Background. The objective of this study was to develop a nomogram that can predict lymph node metastasis (LNM) in patients with cervical adenocarcinoma (cervical AC). Methods. A total of 219 patients with cervical AC who had undergone radical hysterectomy and lymphadenopathy between 2005 and 2021 were selected for this study. Both univariate and multivariate logistic regression analyses were performed to analyze the selected key clinicopathologic features and develop a nomogram and underwent internal validation to predict the probability of LNM. Results. Lymphovascular invasion (LVI), tumor   size ≥ 4  cm, and depth of cervical stromal infiltration were independent predictors of LNM in cervical AC. However, the Silva pattern was not found to be a significant predictor in the multivariate model. The Silva pattern was still included in the model based on the improved predictive performance of the model observed in the previous studies. The concordance index ( C -index) of the model increased from 0.786 to 0.794 after the inclusion of the Silva pattern. The Silva pattern was found to be the strongest predictor of LNM among all the pathological factors investigated, with an OR of 4.37 in the nomogram model. The nomogram developed by incorporation of these four predictors performed well in terms of discrimination and calibration capabilities ( C − index = 0.794 ; 95% confidence interval (CI), 0.727–0.862; Brier   score = 0.127 ). Decision curve analysis demonstrated that the nomogram was clinically effective in the prediction of LNM. Conclusion. In this study, a nomogram was developed based on the pathologic features, which helped to screen individuals with a higher risk of occult LNM. As a result, this tool may be specifically useful in the management of individuals with cervical AC and help gynecologists to guide clinical individualized treatment plan.

Circular RNA hsa_circ_0000511 Improves Epithelial Mesenchymal Transition of Cervical Cancer by Regulating hsa-mir-296-5p/HMGA1

As the second largest gynecological cancer, cervical cancer has been widely reported in recent years in which circular RNA is involved in the disease process. We earlier found that the expression of hsa_circ_0000511 in cervical cancer cells increased significantly, but its role in the process of cervical cancer is not clear. The purpose of this study is to explore its possible mechanisms in cervical cancer. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), cell counting kit-8 assay, Transwell test, cell transfection, RNA pull-down assay and dual-luciferase reporter assay, and Western blot analysis were used to detect the expression and distribution of hsa_circ_0000511 in SiHa and HeLa cells, the ability of invasion and proliferation, and the modulated relationships between hsa_circ_0000511 and hsa-mir-296-5p, hsa-mir-296-5p, and HMGA1. hsa_circ_0000511 had the highest expression in SiHa and HeLa cells, and the expression in the cytoplasm was significantly higher than that in the nucleus, and its expression was not affected by RNase R. When hsa_circ_0000511 was silenced, its expression in SiHa and HeLa cells was significantly decreased; the proliferation, invasion, and migration abilities of the two kinds of cells were significantly enhanced; and the protein expression of E-cadherin was significantly upregulated, while the protein expression of N-cadherin was significantly downregulated. The expression of hsa-mir-296-5p was lower in SiHa and HeLa cells; however, its expression was increased when hsa_circ_0000511 was inhibited and decreased when hsa_circ_0000511 was overexpressed, so did the ability of proliferation, invasion, and migration and the protein expression of E-cadherin. Interestingly, the protein expression of HMGA1 also changed in these two cells when hsa-mir-296-5p was inhibited or overexpressed. Our results indicate that the upregulated hsa_circ_0000511 can inhibit the proliferation, invasion, and migration of SiHa and HeLa cells by regulating hsa-mir-296-5p/HMGA1, suggesting that the hsa_circ_0000511/hsa-mir-296-5p/HMGA1 pathway may be a potential target for the treatment of cervical cancer.

Downregulated PRNP Facilitates Cell Proliferation and Invasion and Has Effect on the Immune Regulation in Ovarian Cancer

Background. Ovarian cancer (OC) seriously threatens women’s life. Ferroptosis plays an essential role in the initiation and development of OC. However, more molecular targets and mechanisms for ferroptosis in OC remain to be further elucidated. Methods. Several OC datasets were integrated in this study and three candidate genes including PRNP were further screened out as the ferroptosis-related gene which was differentially expressed in OC. Then, comprehensive evaluations concerning gene expression, clinical implication, in vitro validation of expression and functional experiments, prediction of downstream molecules and related signal pathways, and immune-modulating function were performed. Results. PRNP was the only downregulated ferroptosis-related gene with prognostic value for OC patients. The decreased mRNA and protein expression was verified in OC tissues and cell lines. PRNP was significantly correlated with cancer stages, primary therapy outcomes, and age in OC patients. Moreover, we found that overexpression of PRNP inhibited the proliferation, migration, and invasion ability of OC cells through in vitro experiments. PRNP was enriched to the Ras signaling pathway. PRNP expression was positively correlated with the infiltration of immune cells, such as mast cells, T effector memory cells, plasmacytoid DC cells, NK cells, and eosinophils. In addition, the association of PRNP with other immune signatures was also found. Conclusion. This study demonstrated for the first time showed that ferroptosis-related gene PRNP exerted a tumor suppressive role in OC and the aberrant expression and function of PRNP making it a potential novel biomarker for OC diagnosis, prognosis, and response to immunotherapies.

m6A-Related lncRNA Signature Is Involved in Immunosuppression and Predicts the Patient Prognosis of the Age-Associated Ovarian Cancer

Background. Epithelial ovarian cancers are age-associated diseases, usually diagnosed at an advanced stage. lncRNA has been discovered to interplay with N6-methyladenosine (m6A), working in tandem to promote cancer progression and worsening patient outcomes. This study is aimed at investigating the roles and mechanism of m6A-related lncRNA signature on ovarian cancers. Methods. We retrieved TCGA and CGGA sequencing data to identify m6A-related lncRNA signature and constructed an m6A score (MS) using the LASSO algorithm. A clinical nomogram was then established to predict the overall survival of patients. Subsequently, GSEA analyses were conducted to obtain pathways involved. Expression of HLA genes, 28 tumor-infiltrating lymphocyte infiltration, and anticancer cycle were analyzed the immunological differences between high-MS and low-MS groups. Finally, immune checkpoint gene expressions and IC50 of chemotherapeutic drugs were calculated, and CMap was run to identify the potential compounds and their corresponding mechanisms. Results. We identified 16 m6A-related lncRNAs and constructed an MS model. The high-MS group showed a poor prognosis. A clinical nomogram consists of MS, and age was constructed and predicted the 1-, 3-, and 5-year survival with high accuracy. GSEA analyses presented downregulated antigen processing and presentation pathways. Immunocyte infiltrating analyses demonstrated that high-MS was associated with high infiltration of Treg cells, macrophages, and low Th1/Th2 rate. Also, high expression of immune checkpoint genes NRP1, TNFSF9, and VSIR was observed in the high-MS group. Finally, the high-MS group also predicted low IC50 of vinorelbine and vorinostat. Conclusion. This study constructed a robust prediction model for prognostic management and revealed the cross-talk between m6A and immunosuppression. Besides, the m6A lncRNA signature can predict the chemotherapeutic drug response. These will shed light on the development of novel therapeutic strategies and render survival benefits for ovarian patients.

Anticancer Effect of Puerarin on Ovarian Cancer Progression Contributes to the Tumor Suppressor Gene Expression and Gut Microbiota Modulation

Ovarian cancer (OC) causes more deaths than any other cancer of the female reproductive system due to its late presentation and malignant nature. Although significant progress has been made in the diagnosis and treatment of OC over the last decade, chemotherapeutic drug resistance and cancer recurrence remain serious challenges in OC management. In the field of cancer therapy, traditional Chinese herbal medicines and their active compounds have been widely reported to have favorable therapeutic effects on cancer. Recent studies have also revealed the protective effect of puerarin in cancer, but the exact role and underlying mechanism of puerarin in OC remain unclear. Here, we established in vivo and in vitro OC models to evaluate the anticancer effect of puerarin. It was found that puerarin significantly inhibited OC cell viability and proliferation and induced cell apoptosis. In OC model mice, puerarin treatment suppressed tumor formation and modulated the gut microbiome. In addition, the expression of tumor suppressor genes was activated by puerarin in vitro and in vivo. These findings add to the existing knowledge on the usefulness of herbal active ingredients for the prevention and treatment of OC and provide a new perspective regarding the therapeutic potential of puerarin in cancer.

Construction of Metabolic Molecular Classification and Immune Characteristics for the Prognosis Prediction of Ovarian Cancer

Background. Ovarian cancer (OC) is a malignant tumor that seriously threatens women’s health. Molecular classification based on metabolic genes can reflect the deeper characteristics of ovarian cancer and provide support for prognostic evaluation and the guidance of individualized treatment. Method. The metabolic subtypes were determined by consensus clustering and CDF. We used the ssGSEA method to calculate the IFNγ score of each patient. The CIBERSORT method was used to evaluate the score distribution and differential expression of 22 immune cells, and LDA was applied to establish a subtype classification feature index. The Kaplan-Meier and ROC curves were generated to validate the prognostic performance of metabolic subtypes in different cohorts. WGCNA was used to screen the coexpression modules associated with metabolic genes. Results. We obtained three metabolic subtypes (MC1, MC2, and MC3). MC2 had the best prognosis, and MC1 and MC3 had poor prognoses. Consistently, MC2 subtype had higher T cell lytic activity and lower angiogenesis, IFNγ, T cell dysfunction, and rejection scores. TIDE analysis showed that MC2 patients were more likely to benefit from immunotherapy; MC1 patients were more sensitive to immune checkpoint inhibitors and traditional chemotherapy drugs. The multiclass AUCs based on the RNASeq and GSE cohorts were 0.93 and 0.84, respectively. Finally, we screened 11 potential gene markers related to the metabolic characteristic index that could be used to indicate the prognosis of OC. Conclusion. Molecular subtypes related to metabolism are crucial to comprehensively understand the molecular pathological characteristics related to metabolism for OC development, explore reliable markers for prognosis, improve the OC staging system, and guide personalized treatment.

An Integrative Analysis Revealing ZFHX4-AS1 as a Novel Prognostic Biomarker Correlated with Immune Infiltrates in Ovarian Cancer

Ovarian cancer (OC) is the main cause of deaths worldwide in female reproductive system malignancies. Growing studies have indicated that eRNAs could regulate cellular activities in various tumors. Yet the potential roles of eRNAs in OC progression have not been elucidated. Thus, comprehensive assays were needed to screen the critical eRNAs and to explore their possible function in OC. We used Kaplan–Meier methods to identify survival-associated eRNAs in OC based on TCGA datasets. The levels of ZFHX4-AS1 were examined using TCGA datasets. Further exploration was carried out based on the following assays: clinical and survival assays, GO terms, and KEGG assays. TIMER was applied to delve into the relationships between ZFHX4-AS1 and tumor immune infiltration. In this research, we observed 71 survival-related eRNAs in OC patients. ZFHX4-AS1 was highly expressed in OC specimens and predicted a poor prognosis of OC patients. In addition, high ZFHX4-AS1 expression was positively related to the advanced stages of OC specimens. Multivariate assays revealed that ZFHX4-AS1 was an independent prognostic factor for overall survival of OC patients. KEGG analysis indicated that ZFHX4-AS1 may play a regulatory effect on TGF-beta signaling, PI3K-Akt signaling, and proteoglycans in cancer. The pan-cancer validation indicated that ZFHX4-AS1 was related to survival in eight tumors, namely, UCEC, STAD, SARC, OV, ACC, KICH, KIRC, and BLCA. The expression of ZFHX4-AS1 was correlated with the levels of B cells, T cell CD8+, neutrophil, macrophage, and myeloid dendritic cells. Simultaneously, ZFHX4-AS1 may be a prognostic biomarker and a distinctly immunotherapy-related eRNA in OC.

HIF-1α Regulated WTAP Overexpression Promoting the Warburg Effect of Ovarian Cancer by m6A-Dependent Manner

N6-methyladenosine (m6A) RNA methylation has been determined to execute crucial functions in tumorigenesis and cancer development. WT1-associated protein (WTAP) has an important “writer” role in m6A modification, and it is also a nuclear protein that colocalizes with splicing factors and plays a critical role in cell function and cancer progression. However, little is known about the role of WTAP in ovarian cancer (OC) and its mechanisms. In this study, we found for the first time that hypoxia-inducible factor (HIF)-1α could positively regulate increased expression of WTAP under hypoxia. And further results revealed that WTAP expression was closely associated with the clinicopathological features of OC, and high expression of WTAP predicted low survival rate in patients with OC. In addition, cell proliferation and invasive capacity were significantly reduced after knockdown of WTAP expression in OC cells. However, cell proliferation and invasive ability were significantly enhanced after overexpression of WTAP. Additionally, we find that WTAP interacts with DGCR8 (a crucial chip protein) to regulate the expression of microRNA-200 (miR-200) in an m6A-dependent way. Further experiments showed that the key glycolysis enzyme HK2 could be positively regulated by miR-200, which significantly affected the intracellular Warburg effect. In conclusion, this is considered uncovered that upregulation of WTAP expression by HIF-1α intercedes with miRNA processing, accelerates the Warburg impact, and advances the event and advancement of tumor, thus giving a novel viewpoint on m6A adjustment in OC movement.

TILs and Anti‐PD1 Therapy: An Alternative Combination Therapy for PDL1 Negative Metastatic Cervical Cancer

Background . We investigated the efficacy of TILs and anti‐PD1 combination therapy in patients with metastatic cervical cancer with low MSI expression and PDL1‐negative. Methods . A total of 80 patients were put on TILs and anti‐PD1 combination therapy, and the progression‐free survival time (PFS) and overall survival time (OS) were assessed by Kaplan–Meier analysis. Univariate and multivariate analyses were performed to identify factors that could predict the prognosis of metastatic cervical cancer in the previously described patients. Results . The objective response rate was 25%, whereas the mPFS and mOS were 6.1 and 11.3 months, respectively. The therapeutic efficacy was influenced by the characteristics of TILs, infection with HPV, and development of fever just after the therapy. Conclusion . Overall, our results show that the combination therapy of TILs and anti‐PD1 significantly improves the prognosis of metastatic cervical cancer.

Genome‐Wide Profiling of Human Papillomavirus DNA Integration into Human Genome and Its Influence on PD‐L1 Expression in Chinese Uygur Cervical Cancer Women

Background . The Uygur is the fifth most populous ethnic group in China. Compared to other Chinese population, cervical cancer in them had high incidence, and HPV infection also was particular. Their HPV integration situation has never been reported. We aimed to investigate the integration situation of 20 subtypes of HPV gene into host cell genome in Chinese Uygur cervical cancer patients; meanwhile, we explored the influence of gene integration on PD‐L1 expression. Methods . 40 frozen Chinese Uygur cervical cancer specimens with positive HPV infection were obtained from the cancer prevention and treatment institute of Tumor Hospital Affiliated to Xinjiang Medical University. The integration situation of HPV gene into host cell genome was detected by Agilent SureSelect™ Target Enrichment Chip and Next‐Generation Sequencing. The related genes were analyzed by GO functional annotation and KEGG pathway enrichment. The expression levels of PD‐L1 in cancer cells were tested by immunohistochemical assay (IHC). Meanwhile, the relationship between PD‐L1 levels in cancer cells and gene integration were analyzed. Results . The HPV multiple infection rate by HIVID was as high as 92.5%, much higher than 35.0% by the commercial kit ( P < 0.05). There were 13423 integration events in 40 specimens, involving 6867 human genes. These integration events were distributed on all human chromosomes, and chromosome 19 had the excessive concentration phenomenon of integration events. There were some integration hotspots in human genome such as PPP1R37, HECW2, EMBP1, ANKRD50, SPTBN4, LINC00895, LYRM4‐AS1, LINC00374, RBFOX1, CSMD1, CDH13, and KLHL4. Insertion breakpoints can be found in all gene regions of the HPV genome. The actual observation of the integration times of E1 and E6 was much higher than the expected value, while the actual observation times of E5 were much lower than the expected value. The result of GO functional analysis showed that binding molecular function and cellular process biological process were the main ways to influence the cell biological behavior of HPV gene integration. The enrichment pathway analysis of KEGG showed that pathways in cancer were the most important enrichment pathways involved in the genomic integration of HPV. The positive PD‐L1 rate was 62.5%. Logistic regression analysis showed that 9p24.1 existing integration sites and the number of all gene integration were risk factors for PD‐L1 expression (odds ratio 17.313 and 1.012; 95% confidence interval 1.691‐177.213 and 1.001‐1.023). Conclusions and Relevance . Most high‐frequency sites of HPV integration in Chinese Uygur cervical cancer are related to cancer progression, and the gene integration hotspots may be potential HPV carcinogenic targets. The problem of multiple HPV infection in Chinese Uygur cervical cancer patients should be paid attention. L1 and E6 genes are inapposite as the target gene of commercial HPV type detection kit, because of high‐frequency breakpoints in these genes. The gene integration especially the integration existing on 9p24.1 could affect the expression level of PD‐L1.

The Immunotherapeutic Effect of SIRP α ‐Silenced DCs against Cervical Cancer

Signal regulatory protein α (SIRP α ), a transmembrane protein that is predominantly expressed in dendritic cells (DCs) or macrophages, interacts with CD47 that is overexpressed in almost all types of tumor cells. The interaction between SIRP α and CD47 leads to a negative signal that prevents the phenotypic and functional maturation of DC and inhibits phagocytosis. The SIRP α knockdown in DCs that were pulsed with a modified HPV16E7 (HPV16mE7) protein with enhanced antigenicity and reduced transformation activity results in increased cytokine (TNF‐ α /IL‐12/IL‐6) secretion, IFN‐ γ secretion by T lymphocytes, and in vitro / in vivo tumoricidal activity against cervical cancer cells. Taken together, these results suggest that SIRP α ‐silenced DC vaccination presented potential therapeutic implications against cervical cancer.

Expression and Significance of Immune Checkpoints in Clear Cell Carcinoma of the Uterine Cervix

The purpose of this study was to investigate the expression levels of the immune checkpoint proteins, programmed cell death‐ligand 1 (PD‐L1), B7‐H3, B7‐H4, and V‐domain Ig suppressor of T cell activation (VISTA), as well as the significance thereof, in clear cell carcinoma (CCC) of the cervix (a rare histological subtype of cervical cancer). We also compared the expression statuses of these biomarkers in cervical CCCs with those in cervical squamous cell carcinomas (SCCs). We evaluated the expression of PD‐L1, B7‐H3, B7‐H4, and VISTA in 50 cervical CCCs and 100 SCCs using immunohistochemical staining and investigated the associations between these markers, clinicopathologic features, and survival in patients with CCCs. Of the cervical CCC samples examined, 22%, 16%, 32%, and 34% were positive for PD‐L1, B7‐H3, B7‐H4, and VISTA, respectively. Nineteen samples (38%) were negative for all 4 of these markers, whereas 31 (62%) expressed at least 1 marker. None of these markers was associated with the investigated clinicopathologic variables or patient survival. PD‐L1, B7‐H3, and VISTA were observed significantly more frequently in SCCs than in CCCs of the cervix. Our study confirmed the expression of immune checkpoint proteins in cervical CCCs and indicated their nonredundant and complementary roles. As such, our data suggest that monotherapeutic immune checkpoint blockade may not be sufficiently effective in patients with cervical CCC.

TNPO1-Mediated Nuclear Import of FUBP1 Contributes to Tumor Immune Evasion by Increasing NRP1 Expression in Cervical Cancer

Far upstream element binding protein 1 (FUBP1), a DNA-binding protein, participates in diverse tumor-promoting behaviors by regulating the expression of oncogenes in the nucleus, but the underlying mechanisms remain to be elucidated. In the present study, we found that FUBP1 mRNA and protein expressions were markedly upregulated and closely linked with poor prognosis in cervical cancer. In vitro, functional experiments showed that knockdown of FUBP1 inhibited CC cell proliferation and migration. Therefore, FUBP1 plays a prooncogenic function in CC progression. Further investigations for the first time demonstrated that nuclear localization of FUBP1 regulated the gene expression of immune checkpoint NRP1. Moreover, our work demonstrated that FUBP1 translocated into the nucleus which was mediated by interacting with Transportin-1 (TNPO1). Collectively, this study revealed that FUBP1 might be a potential therapeutic target for the restriction of tumor progression.

Activation of FGD5-AS1 Promotes Progression of Cervical Cancer through Regulating BST2 to Inhibit Macrophage M1 Polarization

Accumulating evidence has elucidated the biological function of lncRNAs in various tumors. FGD5 antisense RNA 1 (FGD5-AS1) is identified as a significant tumor regulator in malignancies. Up to now, the detailed function of FGD5-AS1 in cervical cancer and its underlying molecular mechanisms remain uninvestigated. Bone marrow stromal cell antigen 2 (BST2) can play critical roles in immune response, and the roles of BST2 in cervical cancer was explored currently. The level of FGD5-AS1 and BST2 was detected by qRT-PCR in cervical cancer cells. FGD5-AS1 and BST2 expression was significantly upregulated in cervical cancer cells. Then, the decrease of FGD5-AS1 greatly repressed cervical cancer cell growth in vitro. In addition, FGD5-AS1 silencing repressed BST2 expression and suppressed M2 macrophage polarization. Mechanistically, we confirmed that FGD5-AS1 sponged miR-129-5p to reduce its inhibition on BST2. Furthermore, lack of BST2 depressed cervical cancer cell growth, while inducing apoptosis. Loss of BST2 induced M1 macrophage polarization while blocking M2 macrophage polarization. For another, we demonstrated that FGD5-AS1-triggered M2 macrophage polarization was remarkably reversed by miR-129-5p via suppressing BST2. In conclusion, FGD5-AS1 induced M2 macrophage polarization via sponging miR-129-5p and modulating BST2, thus contributing to cervical cancer development. Our findings revealed FGD5-AS1/miR-129-5p/BST2 as a new potential target for cervical cancer.

LINC00707 Regulates miR-382-5p/VEGFA Pathway to Enhance Cervical Cancer Progression

Long noncoding RNAs (LncRNAs) are reported to exhibit crucial roles in cancer progression. LINC00707 is recently indicated to be a significant oncogene in various cancers. Up to now, the mechanism of LINC00707 in cervical cancer is still unclear. In this study, our present work was designed to study the biological effects of LINC00707 in cervical cancer. Firstly, the expression level of LINC00707 in cervical cancer was tested. We observed LINC00707 expression was greatly increased in cervical cancer. Then, we assessed the detailed effect of LINC00707 on the development of cervical cancer using CCK-8 assay, Transwell assays, and tumor xenograft experiments. Gain-of-function and loss-of-function assays revealed the function of LINC00707 in cervical cancer progression. In addition, the action of LINC00707 in cervical cancer cells was studied using bioinformatic tools and luciferase reporter experiment. It was displayed that loss of LINC00707 significantly repressed cell growth of cervical cancer. Meanwhile, restoration of LINC00707 expression obviously induced cervical cancer cell growth. Then, we predicted that LINC00707 could serve as a molecular sponge for miR-382-5p to modulate VEGFA expression in cervical cancer. Subsequently, lack of VEGFA expression reversed the influence of miR-382-5p knockdown on cervical cancer cells. In conclusion, our findings evidenced the significant role of LINC00707-miR-382-5p-VEGFA network in cervical cancer and it can provide an attractive target.

CDK12 Promotes Cervical Cancer Progression through Enhancing Macrophage Infiltration

Cervical cancer (CC) is a commonly diagnosed and primary consideration of cancer patient death in female reproductive system malignancy. Cyclin-dependent kinase 12 (CDK12), as a transcription-associated CDK, plays important roles in tumor-promoting behaviors, whereas the underlying mechanisms of CDK12 in CC progression are still obscure. In this report, we investigated the role of CDK12 in cervical cancer. The current study identified CDK12 mRNA and protein expression remarkably upregulated in CC patients. Upregulated CDK12 was closely associated with CC progression and poor prognosis. In vitro and in vivo functional experiments showed that knockdown of CDK12 inhibited cancer cell proliferation and colony formation and promoted apoptosis. Further investigations demonstrated that CDK12 regulated the immune microenvironment to facilitate the progression of CC cells by promoting macrophage infiltration. Meanwhile, we first demonstrated that nuclear import of CDK12 is mediated by TNPO1 and might be a new therapeutic target in oncology. Collectively, this study pointed out the potential of CDK12 to serve as a novel therapeutic target in restricting CC proliferation and cell cycle process through promoting macrophage infiltration.

A Network Pharmacology Study on the Cervix Prescription for Treatment of Cervical Cancer

Purpose. Based on the method of network pharmacology to explore the mechanism of the cervical prescription (CP) in the treatment of cervical cancer (CC). Methods. We obtained the active ingredients and potential targets in the CP from the literature and the systematic pharmacological analysis platform of traditional Chinese medicine (BATMAN-TCM); the database was used to search for targets related to cervical cancer and to map CP and targets; the core targets were screened, and the protein-protein interaction network (PPI) was constructed using the TCM compound-target network and STRING database. Gene ontology (GO) and Kyoto Gene and Genome Encyclopedia (KEGG) pathway enrichment analysis of overlapping targets were performed using DAVID 6.8 online tool. Results. The CP contains 2 active ingredients, corresponding to 301 nonreactive targets; 10 GO biological process related items and 73 signal pathways were obtained. Cell experiments confirmed that the medicated serum of CP could effectively inhibit the proliferation and invasion ability of Hela cells. Conclusion. This study provides valuable information for TCM researchers and clinicians to better understand the main therapeutic targets and therapeutic roles of herbal decoctions in clinical settings. The results of our study preliminarily clarified that the cervical prescription has an inhibitory effect on cervical cancer cells.

HIF-3α-Induced miR-630 Expression Promotes Cancer Hallmarks in Cervical Cancer Cells by Forming a Positive Feedback Loop

Purpose. Hypoxia has crucial functions in the development and metastasis of cervical cancer by inducing the expression of numerous genes, including microRNA genes. But we know little about how the hypoxia factors and microRNAs orchestrate to regulate hallmarks of cervical cancer cells. Methods. We conducted RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) experiments to investigate the targets of HIF-3α or miR-630. ChIP-qPCR and RT-qPCR were carried out to validate the results of ChIP-seq and RNA-seq. Cellular, molecular, and radiation experiments were conducted to explore the functions of miR-630. Results. In this study, we showed that hypoxia-induced overexpression of HIF-3α increased the expression of dozens of miRNAs, including miR-630. Hypoxia could also directly induce miR-630 expression. ChIP-seq data showed that HIF-3α activates miR-630 expression by directly binding to the promoter of its host gene. Meanwhile, stable overexpression of miR-630 increased the expression of HIF-3α, but repressed the expression of HIF-1α, indicating a positive feedback loop between HIF-3α and miR-630. Consequently, stable overexpression of miR-630 in HeLa cells promotes cancer hallmarks, including radioresistance, inhibition of apoptosis, increased migration and invasion, and EMT-mediated metastasis. Meanwhile, inhibition of miR-630 showed opposite features. Conclusion. Taken together, our findings indicate a novel hypoxia-induced HIF-3α and miR-630 regulatory feedback loop contributing to metastasis and progression of cervical cancer cells and suggest that HIF-3α and miR-630 might act as potential biomarkers and therapeutic targets for cervical cancer in the future.

Vaginal Microbial Environment Skews Macrophage Polarization and Contributes to Cervical Cancer Development

As a common female reproductive system malignancy, cervical cancer (CC) disturbs numerous women’s health. This study demonstrates the role of the vaginal microbial environment (Peptostreptococcus anaerobius) in cervical cancer. Functional assays, including cell proliferation assay, tube formation assay, and immunofluorescence staining, revealed the effect of Peptostreptococcus anaerobius-treated macrophages on cell proliferation and the angiogenesis process. The tube formation assay disclosed the function of Peptostreptococcus anaerobius-treated macrophages on angiogenesis. In vivo assays were also established to explore the impact of Peptostreptococcus anaerobius-treated macrophages on tumor migration. The results revealed that Peptostreptococcus anaerobius-induced macrophages boosted cervical cancer migration and angiogenesis both in vitro and in vivo. Then, this study unveiled that Peptostreptococcus anaerobius-induced macrophage secreted VEGF to stimulate the angiogenesis in cervical cancer. As a whole, Peptostreptococcus anaerobius-induced macrophage facilitates cervical cancer development through modulation of VEGF expression.

Prognosis Analysis and Validation of Fatty Acid Metabolism-Related lncRNAs and Tumor Immune Microenvironment in Cervical Cancer

Cervical cancer (CC) is the third most common carcinoma and the fourth leading cause of cancer-associated mortality in women. The deregulation of fatty acid metabolism plays a crucial role in the progression of various tumors. This study is aimed at exploring the prognostic values of fatty acid metabolism- (FAM-) related long noncoding RNAs (lncRNAs) in CC. FAM-related differentially expressed genes (DEGs) and lncRNAs were screened in CC specimens based on TCGA datasets. Univariate analysis was carried out on differentially expressed lncRNAs to screen the survival-related lncRNAs. Multivariate assays were performed on the resulting lncRNAs to create a novel risk model. Survival assays were applied to examine the prognostic abilities of our model. Receiver operating characteristic (ROC) analysis was used to evaluate the accuracy of the new model. The association between risk model and immune responses was analyzed. In this study, we screened 9 differently expressed lncRNAs associated with the clinical outcome of CC patients. A nine-lncRNA signature comprising SCAT1, AC119427.1, AC009097.2, MIR100HG, AC010996.1, AL583856.2, MIAT, AP003774.2, and AC004540.2 was established to predict overall survival of CC. Survival assays revealed that patients’ high risk score showed a shorter overall survival than those with low risk score. Multivariate assays demonstrated that the nine-gene signature was an independent prognostic factor in CC. In addition, we observed that APC_co_stimulation, CCR, and parainflammation were distinctly different between low-risk and high-risk groups. Our group observed a distinct difference in the expressions of CD44, TNFRSF8, CD276, LAG3, TNFRSF14, TMIGD2, VTCN1, TNFRSF25, CD80, NRP1, TNFRSF18, CD70, TNFSF9, and LGALS9 between the two groups of patients. Overall, our findings indicated that the 9 FAM-related lncRNA signature might be a promising prognostic factor for CC and can promote the management of FAM-related therapy in clinical practice.

Systematic Construction and Validation of a Novel Ferroptosis-Related Gene Model for Predicting Prognosis in Cervical Cancer

Background and Purpose. Ferroptosis, a mechanism of cell death that is iron-dependent, participates in various pathologies of cancer (CC). Nevertheless, the specific function that ferroptosis plays in the onset and progression of cervical cancer (CC) is yet uncertain. This research sought to examine the value of ferroptosis-related genes (FRGs) in the progression and prognosis of CC. Methods. Datasets containing RNA sequencing and corresponding clinical data of cervical cancer patients were obtained from searching publicly accessible databases. The “NMF” R package was conducted to calculate the matrix of the screened prognosis gene expression. Ferroptosis-related differential genes in cervical cancer were detected using the “limma” R function and WGCNA. The least absolute shrinkage and selection operator (LASSO) algorithm and Cox regression analysis were conducted to develop a novel prognostic signature. The prediction model was verified by the nomogram integrating clinical characteristics; the GSE44001 dataset was used as an external verification. Then, the immune status and tumor mutation load were explored. Finally, immunohistochemistry as well as quantitative polymerase chain reaction (RT-qPCR) was utilized to ascertain the expression of FRGs. Results. Two molecular subgroups (cluster 1 and cluster 2) with different FRG expression patterns were recognized. A ferroptosis-related model based on 4 genes (VEGFA, CA9, DERL3, and RNF130) was developed through TCGA database to identify the unfavorable prognosis cases. Patients in cluster 1 showed significantly decreased overall survival in contrast with those in cluster 2 ( P < 0.05 ). The LASSO technique and Cox regression analysis were both utilized to establish the independence of the prognostic model. The validity of nomogram prognostic predictions has been well demonstrated for 3- and 5-year survival in both internal and external data validation cohorts. These two subgroups showed striking differences in tumor-infiltrating leukocytes and tumor mutation burden. The low-risk subgroup showed a longer overall survival time with a higher immune cell score and higher tumor mutation rate. Gene functional enrichment analyses revealed predominant enrichment in various tumor-associated signaling pathways. Finally, the expression of each gene was confirmed by immunohistochemistry and RT-qPCR. Conclusion. A novel and comprehensive ferroptosis-related gene model was proposed for cervical cancer which was capable of distinguishing the patients independently with high risk for poor survival, and targeting ferroptosis may represent a promising approach for the treatment of CC.

PMEPA1 Serves as a Prognostic Biomarker and Correlates with Immune Infiltrates in Cervical Cancer

Emerging studies have demonstrated that Prostate transmembrane protein androgen induced 1 (PMEPA1) plays crucial roles in the carcinogenesis of many developing human tumors. However, the clinical significance of PMEPA1 expression in cervical cancer (CC) and its contribution to cancer immunity have not been investigated. In this study, we identified PMEPA1 as a survival-related gene in CC based on TCGA datasets. Univariate and multivariate analysis showed that PMEPA1 expression was an independent predictor for overall survival in CC patients. We could observe a strong negative correlation between PMEPA1 expression and PMEPA1 methylation. Two CpG sites of PMEPA1 were associated with overall survival, and one CpG site of PMEPA1 was associated with progression-free survival. The low level of PMEPA1 methylation was associated with advanced clinical stage of CC patients. KEGG assays revealed the genes associated with PMEPA1 expression were mainly enriched in several tumor-related pathways. Increased PMEPA1 expressions were observed to be positively related to high immune infiltration levels in several immune cells. Finally, the pan-cancer assays revealed that PMEPA1 expression was associated with the overall survival of UVM, PAAD, LUSC, BLCA, CESC, and LUAD. Taken together, PMEPA1 is a prognosis-related biomarker for multiple cancer types, especially CC. PMEPA1 is involved in tumor immunity, suggesting PMEPA1 may be a potential immunotherapeutic target in CC.

Concurrent Chemoradiotherapy Increases the Levels of Soluble Immune Checkpoint Proteins in Patients with Locally Advanced Cervical Cancer

Purpose. Concurrent chemoradiotherapy (CCRT) has been widely applied to locally advanced cervical cancer (LACC) patients, inducing the massive release of antigen and systematic immunomodulatory effects. However, its effect on the soluble immune checkpoint proteins (sICPs) remains unclear, which might play a key role in the immune response. Therefore, the current study explored changes in the levels of 16 sICPs in LACC patients during CCRT. Methods. We prospectively enrolled fifty-one LACC patients treated with CCRT and collected patients’ blood before, during and after CCRT. The levels of 16 sICPs were measured using the Luminex platform, and the changes were measured using Friedman test with Bonferroni’s posttest. One month after CCRT, the tumor response was evaluated according to the RECIST 1.1 guidelines. Results. The levels of soluble T-cell immunoglobulin and mucin-domain containing-3 (sTIM-3) significantly increased during CCRT ( P = 0.041 ), while those of the soluble B and T lymphocyte attenuator (sBTLA), sCD40, soluble glucocorticoid-induced tumor necrosis factor receptor ligand (sGITRL), sCD80, sCD86, sPD-1, sPD-L1, sCTLA-4, and soluble inducible T-cell costimulator (sICOS) significantly increased after CCRT (all P < 0.05 ). Other sICPs showed no significant changes throughout the CCRT (all P > 0.05 ). 41 (80%), 8 (16%), and 2 (4%) patients showed complete response (CR), partial response (PR), and stable disease (SD) after CCRT, respectively. Interestingly, the level of soluble lymphocyte-activation gene 3 (sLAG-3) was significantly higher among the PR/SD patients as compared to the CR after CCRT ( P = 0.009 ). Conclusions. This study revealed that CCRT might elevate the serum levels of sTIM-3, sBTLA, sCD40, sGITRL, sCD80, sCD86, sPD-1, sPD-L1, sCTLA-4, and sICOS in the patients with LACC. The sLAG-3 level was higher in the patients with poor response to CCRT. These findings revealed the dynamic changes in the sICPs levels during CCRT, which might be helpful in designing optimal treatment strategies for LACC patients.