Activation of AMPK inhibits cervical cancer growth by hyperacetylation of H3K9 through PCAF.

BACKGROUND: Dysregulation in histone acetylation, a significant epigenetic alteration closely associated with major pathologies including cancer, promotes tumorigenesis, inactivating tumor-suppressor genes and activating oncogenic pathways. AMP-activated protein kinase (AMPK) is a cellular energy sensor that regulates a multitude of biological processes. Although a number of studies have identified the mechanisms by which AMPK regulates cancer growth, the underlying epigenetic mechanisms remain unknown. METHODS: The impact of metformin, an AMPK activator, on cervical cancer was evaluated through assessments of cell viability, tumor xenograft model, pan-acetylation analysis, and the role of the AMPK-PCAF-H3K9ac signaling pathway. Using label-free quantitative acetylproteomics and chromatin immunoprecipitation-sequencing (ChIP) technology, the activation of AMPK-induced H3K9 acetylation was further investigated. RESULTS: In this study, we found that metformin, acting as an AMPK agonist, activates AMPK, thereby inhibiting the proliferation of cervical cancer both in vitro and in vivo. Mechanistically, AMPK activation induces H3K9 acetylation at epigenetic level, leading to chromatin remodeling in cervical cancer. This also enhances the binding of H3K9ac to the promoter regions of multiple tumor suppressor genes, thereby promoting their transcriptional activation. Furthermore, the absence of PCAF renders AMPK activation incapable of inducing H3K9 acetylation. CONCLUSIONS: In conclusion, our findings demonstrate that AMPK mediates the inhibition of cervical cancer growth through PCAF-dependent H3K9 acetylation. This discovery not only facilitates the clinical application of metformin but also underscores the essential role of PCAF in AMPK activation-induced H3K9 hyperacetylation.

M2 macrophages regulate KDM6B/PFKFB2 metabolic reprogramming of cervical squamous cell carcinoma through CXCL1.

Macrophages in the tumor microenvironment can polarize into M1 or M2 forms, with M2 macrophages (M2φ) promoting tumor growth and metastasis in cervical squamous cell carcinoma (CESC). This study explored the effects of M2φ on CESC metabolic reprogramming both in vitro and in vivo. Results showed that M2φ secreted CXCL1, which significantly increased CESC migration and metabolic regulation. Further experiments revealed that CXCL1 upregulated KDM6B to enhance PFKFB2 transcriptional activity, thus regulating CESC glucose metabolism. Transcriptome sequencing screened 5 upregulated genes related to glycolysis, with PFKFB2 showing the most significant increase in cells treated with rCXCL1. Dual-luciferase reporter assay confirmed that rCXCL1 enhances PFKFB2 transcriptional activity. Bioinformatics analysis revealed a high correlation between expressions of KDM6B and PFKFB2 in CESC. Mechanistic experiments demonstrated that KDM6B inhibited H3K27me3 modification to activate PFKFB2 transcriptional expression. In conclusion, M2φ secreted CXCL1 to promote CESC cell migration and invasion, and CXCL1 activated KDM6B expression in CESC cells, inhibiting H3K27 protein methylation modification, and enhanced PFKFB2 transcriptional activity to regulate CESC glucose metabolism. These results provided new insights into the complex interplay between the immune system and cancer metabolism, which may have broader implications for understanding and treating other types of cancer.

Chemoradiotherapy-induced ACKR2+ tumor cells drive CD8+ T cell senescence and cervical cancer recurrence.

Tumor recurrence after chemoradiotherapy is challenging to overcome, and approaches to predict the recurrence remain elusive. Here, human cervical cancer tissues before and after concurrent chemoradiotherapy (CCRT) analyzed by single-cell RNA sequencing reveal that CCRT specifically promotes CD8+ T cell senescence, driven by atypical chemokine receptor 2 (ACKR2)+ CCRT-resistant tumor cells. Mechanistically, ACKR2 expression is increased in response to CCRT and is also upregulated through the ligation of CC chemokines that are produced by activated myeloid and T cells. Subsequently, ACKR2+ tumor cells are induced to produce transforming growth factor ß to drive CD8+ T cell senescence, thereby compromising antitumor immunity. Moreover, retrospective analysis reveals that ACKR2 expression and CD8+ T cell senescence are enhanced in patients with cervical cancer who experienced recurrence after CCRT, indicating poor prognosis. Overall, we identify a subpopulation of CCRT-resistant ACKR2+ tumor cells driving CD8+ T cell senescence and tumor recurrence and highlight the prognostic value of ACKR2 and CD8+ T cell senescence for chemoradiotherapy recurrence.

Mesencephalic astrocyte-derived neurotrophic factor inhibits cervical cancer progression via regulating macrophage phenotype.

BACKGROUND: Cervical cancer is a common gynecologic malignant tumor, but the critical factors affecting cervical cancer progression are still not well demonstrated. Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been widely recognized as an anti-inflammatory factor to regulate macrophage polarization. In this study, the effect and mechanism of MANF on cervical cancer were preliminarily explored. METHODS AND RESULTS: Kaplan-Meier curve was used to show the overall survival time of the involved cervical cancer patients with high and low MANF expression in cervical cancer tissues. MANF was highly expressed in peritumoral tissues of cervical carcinoma by using immunohistochemistry and western blot. MANF mRNA level was detected by using qRT-PCR. Dual-labeled immunofluorescence showed MANF was mainly expressed in macrophages of cervical peritumoral tissues. Moreover, MANF-silenced macrophages promoted HeLa and SiHa cells survival, migration, invasion and EMT via NF-κB signaling activation. The results of tumor formation in nude mice indicated MANF-silenced macrophages promoted cervical tumor formation in vivo. CONCLUSION: Our study reveals an inhibitory role of MANF in cervical cancer progression, indicating MANF as a new and valuable therapeutic target for cervical cancer treatment.

TPP1 is associated with risk of advanced precursors and cervical cancer survival.

It is unclear how telomere-binding protein TPP1 interacts with human telomerase reverse transcriptase (hTERT) and influences cervical cancer development and progression. This study included all eligible 156 cervical cancers diagnosed during 2003-2008 and followed up through 2014, 102 cervical intraepithelial neoplasia (CIN) patients, and 16 participants with normal cervix identified at the same period. Correlation of expression of TPP1 and hTERT in these lesions was assessed using Kappa statistics. TPP1 was knocked down by siRNA in three cervical cancer cell lines. We assessed mRNA expression using quantitative real-time polymerase chain reaction and protein expression using tissue microarray-based immunohistochemical staining. We further analyzed the impact of TPP1 expression on the overall survival of cervical cancer patients by calculating the hazard ratio (HR) with 95% confidence intervals (CIs) using the multivariable-adjusted Cox regression model. Compared to the normal cervix, high TPP1expression was significantly associated with CIN 3 and cervical cancers (P<0.001 for both). Expressions of TPP1 and hTERT were highly correlated in CIN 3 (Kappa statistics = 0.50, P = 0.005), squamous cell carcinoma (Kappa statistics = 0.22, P = 0.011), and adenocarcinoma/adenosquamous carcinoma (Kappa statistics = 0.77, P = 0.001). Mechanistically, knockdown of TPP1 inhibited the expression of hTERT in both mRNA and protein levels. High expression of TPP1 (HR = 2.61, 95% CI 1.23-5.51) and co-high expression of TPP1 and hTERT (HR = 2.38, 95% CI 1.28-4.43) were independently associated with worse survival in cervical cancer patients. TPP1 and hTERT expression was correlated and high expression of TPP1 was associated with high risk of CIN 3 and cervical cancer and could predict a worse survival in cervical cancer.

Inhibition of NF-kB and COX-2 by andrographolide regulates the progression of cervical cancer by promoting PTEN expression and suppressing PI3K/AKT signalling pathway.

In the face of recent advances in Cervical cancer (CC) treatment, therapeutic and surgical procedures for CC management are still inadequate. In the current study for the first time Andrographolide (Andro) has been explored for its multitarget therapeutic efficacy on NF-kB, COX-2, and PI3K/AKT expressions together in CC. The expression levels of NF-kB, COX-2, PI3K and PTEN in the CC patient samples, both at mRNA and protein levels have shown significant association with poor survival and increased tumor aggressiveness. The binding efficacy of Andro was investigated using molecular docking and molecular dynamic simulations, and the protein and ligand complex for NF-kB and COX-2 has shown high binding energy. Andro displayed cytotoxicity by impeding the in-vitro proliferation of CC cells. Andro significantly supressed the NF-kB, COX-2, and PI3K expression and enhanced the expression levels of PTEN at protein levels in-vitro. Andro induced apoptosis in a dose dependent manner and significantly inhibited the migration and invasion of CC cells. Andro exhibited similar activity in-vivo and suppressed the CC tumor growth in xenograft C57BL/6 mice model. The anti-tumor activity of Andro, both in-vitro and in-vivo has shown considerable downregulation of NF-kB and COX-2 and induced apoptosis through impeding the PI3K/AKT signalling pathway. These findings from the above study projects, administration of Andro as an effective alternate safe compound to curtail and impede cervical cancer progression.

M6A-induced transcription factor IRF5 contributes to the progression of cervical cancer by upregulating PPP6C.

Cervical cancer (CC) is a gynaecological malignancy tumour that seriously threatens women's health. Recent evidence has identified that interferon regulatory factor 5 (IRF5), a nucleoplasm shuttling protein, is a pivotal transcription factor regulating the growth and metastasis of various human tumours. This study aimed to investigate the function and molecular basis of IRF5 in CC development. IRF5, protein phosphatase 6 catalytic subunit (PPP6C) and methyltransferase-like 3 (METTL3) mRNA levels were evaluated by quantitative real-time (qRT)-polymerase chain reaction (PCR). IRF5, PPP6C, METTL3, B-cell lymphoma 2 and Bax protein levels were detected using western blot. Cell proliferation, migration, invasion, angiogenesis and apoptosis were determined by using colony formation, 5-ethynyl-2'-deoxyuridine (EdU), transwell, tube formation assay and flow cytometry assay, respectively. Glucose uptake and lactate production were measured using commercial kits. Xenograft tumour assay in vivo was used to explore the role of IRF5. After JASPAR predication, binding between IRF5 and PPP6C promoter was verified using chromatin immunoprecipitation and dual-luciferase reporter assays. Moreover, the interaction between METTL3 and IRF5 was verified using methylated RNA immunoprecipitation (MeRIP). IRF5, PPP6C and METTL3 were highly expressed in CC tissues and cells. IRF5 silencing significantly inhibited cell proliferation, migration, invasion, angiogenesis and glycolytic metabolism in CC cells, while induced cell apoptosis. Furthermore, the absence of IRF5 hindered tumour growth in vivo. At the molecular level, IRF5 might bind with PPP6C to positively regulate the expression of PPP6C mRNA. Meanwhile, IRF5 was identified as a downstream target of METTL3-mediated m6A modification. METTL3-mediated m6A modification of mRNA might promote CC malignant progression by regulating PPP6C, which might provide a promising therapeutic target for CC treatment.

Cervical extracellular matrix hydrogel optimizes tumor heterogeneity of cervical squamous cell carcinoma organoids.

Cervical cancer, primarily squamous cell carcinoma, is the most prevalent gynecologic malignancy. Organoids can mimic tumor development in vitro, but current Matrigel inaccurately replicates the tissue-specific microenvironment. This limitation compromises the accurate representation of tumor heterogeneity. We collected para-cancerous cervical tissues from patients diagnosed with cervical squamous cell carcinoma (CSCC) and prepared uterine cervix extracellular matrix (UCEM) hydrogels. Proteomic analysis of UCEM identified several tissue-specific signaling pathways including human papillomavirus, phosphatidylinositol 3-kinase-AKT, and extracellular matrix receptor. Secreted proteins like FLNA, MYH9, HSPA8, and EEF1A1 were present, indicating UCEM successfully maintained cervical proteins. UCEM provided a tailored microenvironment for CSCC organoids, enabling formation and growth while preserving tumorigenic potential. RNA sequencing showed UCEM-organoids exhibited greater similarity to native CSCC and reflected tumor heterogeneity by exhibiting CSCC-associated signaling pathways including virus protein-cytokine, nuclear factor κB, tumor necrosis factor, and oncogenes EGR1, FPR1, and IFI6. Moreover, UCEM-organoids developed chemotherapy resistance. Our research provides insights into advanced organoid technology through native matrix hydrogels.

Exploring the Role of E6 and E7 Oncoproteins in Cervical Oncogenesis through MBD2/3-NuRD Complex Chromatin Remodeling.

BACKGROUND: Cervical cancer is among the highest-ranking types of cancer worldwide, with human papillomavirus (HPV) as the agent driving the malignant process. One aspect of the infection's evolution is given by epigenetic modifications, mainly DNA methylation and chromatin alteration. These processes are guided by several chromatin remodeling complexes, including NuRD. The purpose of this study was to evaluate the genome-wide binding patterns of the NuRD complex components (MBD2 and MBD3) in the presence of active HPV16 E6 and E7 oncogenes and to determine the potential of identified genes through an experimental model to differentiate between cervical precursor lesions, with the aim of establishing their utility as biomarkers. METHODS: The experimental model was built using the CaSki cell line and shRNA for E6 and E7 HPV16 silencing, ChIP-seq, qRT-PCR, and Western blot analyses. Selected genes' expression was also assessed in patients. RESULTS: Several genes have been identified to exhibit altered transcriptional activity due to the influence of HPV16 E6/E7 viral oncogenes acting through the MBD2/MBD3 NuRD complex, linking them to viral infection and cervical oncogenesis. CONCLUSIONS: The impacted genes primarily play roles in governing gene transcription, mRNA processing, and regulation of translation. Understanding these mechanisms offers valuable insights into the process of HPV-induced oncogenesis.

METTL3's role in cervical cancer development through m6A modification.

N6-methylated adenosine (m6A) is a crucial RNA modification in eukaryotes, particularly in cancer. However, its role in cervical cancer (CC) is unclear. We aimed to elucidate the part of m6A in CC by analyzing methyltransferase-like 3 (METTL3) expression, identifying downstream targets, and exploring the underlying mechanism. We assessed METTL3 expression in CC using western blotting, quantitative polymerase chain reaction (qPCR), and immunohistochemistry. In vitro and in vivo experiments examined METTL3's role in CC. We employed RNA sequencing, methylated RNA immunoprecipitation sequencing, qPCR, and RNA immunoprecipitation qPCR to explore METTL3's mechanism in CC. METTL3 expression was upregulated in CC, promoting cell proliferation and metastasis. METTL3 knockdown inhibited human cervical cancer by inactivating AKT/mTOR signaling pathway. METTL3-mediated m6A modification was observed in CC cells, targeting phosphodiesterase 3A (PDE3A). METTL3 catalyzed m6A modification on PDE3A mRNA through YTH domain family protein 3 (YTHDF3). Our study indicated the mechanism of m6A modification in CC and suggested the METTL3/YTHDF3/PDE3A axis as a potential clinical target for CC treatment.

Causal relationship between lipid-lowering drugs and ovarian cancer, cervical cancer: a drug target mendelian randomization study.

BACKGROUND: The causal impact of lipid-lowering drugs on ovarian cancer (OC) and cervical cancer (CC) has received considerable attention, but its causal relationship is still a subject of debate. Hence, the objective of this study is to evaluate the impact of lipid-lowering medications on the occurrence risk of OC and CC through Mendelian randomization (MR) analysis of drug targets. METHODS: This investigation concentrated on the primary targets of lipid-lowering medications, specifically, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) and proprotein convertase kexin 9 (PCSK9). Genetic variations associated with HMGCR and PCSK9 were derived from published genome-wide association study (GWAS) findings to serve as substitutes for HMGCR and PCSK9 inhibitors. Employing a MR approach, an analysis was conducted to scrutinize the impact of inhibitors targeting HMGCR and PCSK9 on the occurrence of OC and CC. Coronary heart disease (CHD) risk was utilized as a positive control, and the primary outcomes encompassed OC and CC. RESULTS: The findings of the study suggest a notable elevation in the risk of OC among patients treated with HMGCR inhibitors (OR [95%CI] = 1.815 [1.316, 2.315], p = 0.019). In contrast, no significant correlation was observed between PCSK9 inhibitors and the occurrence of OC. Additionally, the analysis did not reveal any noteworthy connection between HMGCR inhibitors, PCSK9 inhibitors, and CC. CONCLUSION: HMGCR inhibitors significantly elevate the risk of OC in patients, but their mechanism needs further investigation, and no influence of PCSK9 inhibitors on OC has been observed. There is no significant relationship between HMGCR inhibitors, PCSK9 inhibitors, and CC.

Elucidating the molecular mechanisms of pterostilbene against cervical cancer through an integrated bioinformatics and network pharmacology approach.

Pterostilbene (PTE), a natural phenolic compound, has exhibited promising anticancer properties in the preclinical treatment of cervical cancer (CC). This study aims to comprehensively investigate the potential targets and mechanisms underlying PTE's anticancer effects in CC, thereby providing a theoretical foundation for its future clinical application and development. To accomplish this, we employed a range of methodologies, including network pharmacology, bioinformatics, and computer simulation, with specific techniques such as WGCNA, PPI network construction, ROC curve analysis, KM survival analysis, GO functional enrichment, KEGG pathway enrichment, molecular docking, MDS, and single-gene GSEA. Utilizing eight drug target prediction databases, we have identified a total of 532 potential targets for PTE. By combining CC-related genes from the GeneCards disease database with significant genes derived from WGCNA analysis of the GSE63514 dataset, we obtained 7915 unique CC-related genes. By analyzing the intersection of the 7915 CC-related genes and the 2810 genes that impact overall survival time in CC, we identified 690 genes as crucial for CC. Through the use of a Venn diagram, we discovered 36 overlapping targets shared by PTE and CC. We have constructed a PPI network and identified 9 core candidate targets. ROC and KM curve analyses subsequently revealed IL1B, EGFR, IL1A, JUN, MYC, MMP1, MMP3, and ANXA5 as the key targets modulated by PTE in CC. GO and KEGG pathway enrichment analyses indicated significant enrichment of these key targets, primarily in the MAPK and IL-17 signaling pathways. Molecular docking analysis verified the effective binding of PTE to all nine key targets. MDS results showed that the protein-ligand complex between MMP1 and PTE was the most stable among the nine targets. Additionally, GSEA enrichment analysis suggested a potential link between elevated MMP1 expression and the activation of the IL-17 signaling pathway. In conclusion, our study has identified key targets and uncovered the molecular mechanism behind PTE's anticancer activity in CC, establishing a firm theoretical basis for further exploration of PTE's pharmacological effects in CC therapy.

EPAS1, a hypoxia- and ferroptosis-related gene, promotes malignant behaviour of cervical cancer by ceRNA and super-enhancer.

Hypoxia and Ferroptosis are associated with the malignant behaviour of cervical cancer. Endothelial PAS domain-containing protein 1 (EPAS1) contributes to the progression of cervical cancer. EPAS1 plays important roles in hypoxia and ferroptosis. Using the GEO dataset, machine-learning algorithms were used to screen for hypoxia- and ferroptosis-related genes (HFRGs) in cervical cancer. EPAS1 was identified as the hub gene. qPCR and WB were used to investigate the expression of EPAS1 in normal and cervical cancer tissues. The proliferation, invasion and migration of EPAS1 cells in HeLa and SiHa cell lines were detected using CCK8, transwell and wound healing assays, respectively. Apoptosis was detected by flow cytometry. A dual-luciferase assay was used to analyse the MALAT1-miR-182-5P-EPAS1 mRNA axis and core promoter elements of the super-enhancer. EPAS1 was significantly overexpressed in cervical cancer tissues. EPAS1 could increase the proliferation, invasion, migration of HeLa and SiHa cells and reduce the apoptosis of HeLa and SiHa cell. According to the double-luciferase assay, EPAS1 expression was regulated by the MALAT1-Mir-182-5p-EPAS1 mRNA axis. EPAS1 is associated with super-enhancers. Double-luciferase assay showed that the core elements of the super-enhancer were E1 and E3. EPAS1, an HFRG, is significantly overexpressed in cervical cancer. EPAS1 promotes malignant behaviour of cervical cancer cells. EPAS1 expression is regulated by super-enhancers and the MALAT1-miR-182-5P- EPAS1 mRNA axis. EPAS1 may be a target for the diagnosis and treatment of cervical cancer.

Development and assessment of an RNA editing-based risk model for the prognosis of cervical cancer patients.

RNA editing, as an epigenetic mechanism, exhibits a strong correlation with the occurrence and development of cancers. Nevertheless, few studies have been conducted to investigate the impact of RNA editing on cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC). In order to study the connection between RNA editing and CESC patients' prognoses, we obtained CESC-related information from The Cancer Genome Atlas (TCGA) database and randomly allocated the patients into the training group or testing group. An RNA editing-based risk model for CESC patients was established by Cox regression analysis and least absolute shrinkage and selection operator (LASSO). According to the median score generated by this RNA editing-based risk model, patients were categorized into subgroups with high and low risks. We further constructed the nomogram by risk scores and clinical characteristics and analyzed the impact of RNA editing levels on host gene expression levels and adenosine deaminase acting on RNA. Finally, we also compared the biological functions and pathways of differentially expressed genes (DEGs) between different subgroups by enrichment analysis. In this risk model, we screened out 6 RNA editing sites with significant prognostic value. The constructed nomogram performed well in forecasting patients' prognoses. Furthermore, the level of RNA editing at the prognostic site exhibited a strong correlation with host gene expression. In the high-risk subgroup, we observed multiple biological functions and pathways associated with immune response, cell proliferation, and tumor progression. This study establishes an RNA editing-based risk model that helps forecast patients' prognoses and offers a new understanding of the underlying mechanism of RNA editing in CESC.

Long non-coding RNA NEAT1 promotes aerobic glycolysis and progression of cervical cancer through WNT/ß-catenin/PDK1 axis.

BACKGROUND: Cervical cancer is one of the most common gynecological cancers. Accumulated evidence shows that long non-coding RNAs (lncRNAs) play essential roles in cervical cancer occurrence and progression, but their specific functions and mechanisms remain to be further explored. METHODS: The RT-qPCR assay was used to detect the expression of NEAT1 in cervical cancer tissues and cell lines. CCK-8, colony formation, flow cytometry, western blotting, and Transwell assays were used to evaluate the impact of NEAT1 on the malignant behavior of cervical cancer cells. Glucose consumption, lactate production, ATP levels, ROS levels, MMP levels, and the mRNA expressions of glycolysis-related genes and tricarboxylic acid cycle-related genes were detected to analyze the effect of NEAT1 on metabolism reprograming in cervical cancer cells. The expressions of PDK1, ß-catenin and downstream molecules of the WNT/ß-catenin signaling pathway in cervical cancer cells and tissues were detected by western blotting, RT-qPCR, immunofluorescence and immunohistochemistry assays. RESULTS: This study investigated the role and possible molecular mechanism of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in cervical cancer. Our results showed that NEAT1 was highly expressed in cervical cancer tissues and cell lines. Downregulation of NEAT1 inhibited the proliferation, migration, invasion and glycolysis of cervical cancer cells, while overexpression of NEAT1 led to the opposite effects. Mechanistically, NEAT1 upregulated pyruvate dehydrogenase kinase (PDK1) through the WNT/ß-catenin signaling pathway, which enhanced glycolysis and then facilitated cervical cancer metastasis. Furthermore, NEAT1 maintained the protein stability of ß-catenin but did not affect its mRNA level. We also excluded the direct binding of NEAT1 to the ß-catenin protein via RNA pull-down assay. The suppressive impact of NEAT1 knockdown on cell proliferation, invasion, and migration was rescued by ß-catenin overexpression. The WNT inhibitor iCRT3 attenuated the carcinogenic effect induced by NEAT1 overexpression. CONCLUSION: In summary, these findings indicated that NEAT1 may contribute to the progression of cervical cancer by activating the WNT/ß-catenin/PDK1 signaling axis.

Analysis of the diagnostic performance of PAX1/SOX1 gene methylation in cervical precancerous lesions and its role in triage diagnosis.

Methylation panels, tools for investigating epigenetic changes associated with diseases like cancer, can identify DNA methylation patterns indicative of disease, providing diagnostic or prognostic insights. However, the application of methylation panels focusing on the sex-determining region Y-box 1 (SOX1) and paired box gene 1 (PAX1) genes for diagnosing cervical lesions is under-researched. This study aims to examine the diagnostic performance of PAX1/SOX1 gene methylation as a marker for cervical precancerous lesions and its potential application in triage diagnosis. From September 2022 to April 2023, 181 patients with abnormal HPV-DNA tests or cytological exam results requiring colposcopy were studied at Hubei Maternal and Child Health Hospital, China. Data were collected from colposcopy, cytology, HPV-DNA tests, and PAX1/SOX1 methylation detection. Patients were categorized as control, cervical intraepithelial neoplasia Grade 1 (CIN1), Grade 2 (CIN2), Grade 3 (CIN3), and cervical cancer (CC) groups based on histopathology. We performed HPV testing, liquid-based cytology, and PAX1/SOX1 gene methylation testing. We evaluated the diagnostic value of methylation detection in cervical cancer using DNA methylation positivity rate, sensitivity, specificity, and area under the curve (AUC), and explored its potential for triage diagnosis. PAX1/SOX1 methylation positivity rates were: control 17.1%, CIN1 22.5%, CIN2 100.0%, CIN3 90.0%, and CC 100.0%. The AUC values for PAX1 gene methylation detection in diagnosing CIN1+, CIN2+, and CIN3+ were 0.52 (95% confidence interval [CI]: 0.43-0.62), 0.88 (95% CI: 0.80-0.97), and 0.88 (95% CI: 0.75-1.00), respectively. Corresponding AUC values for SOX1 gene methylation detection were 0.47 (95% CI: 0.40-0.58), 0.80 (95% CI: 0.68-0.93), and 0.92 (95% CI: 0.811-1.00), respectively. In HPV16/18-negative patients, methylation detection showed sensitivity of 32.4% and specificity of 83.7% for CIN1+. For CIN2+ and CIN3+, sensitivity was all 100%, with specificities of 83.0% and 81.1%. Among the patients who underwent colposcopy examination, 166 cases had cytological examination results ≤ASCUS, of which 37 cases were positive for methylation, and the colposcopy referral rate was 22.29%. PAX1/SOX1 gene methylation detection exhibits strong diagnostic efficacy for cervical precancerous lesions and holds significant value in triage diagnosis.

[A multicenter study on the accuracy of PAX1/JAM3 dual genes methylation testing for screening cervical cancer].

Objective: To explore the value of cervical cytologic DNA methylation for screening cervical cancer. Methods: This study was a prospective multicenter study conducted from May to October 2022 in Peking Union Medical College Hospital, Zhejiang Provincial People's Hospital, and the Second Affiliated Hospital of Zhejiang University School of Medicine. Women who accepted opportunistic cervical cancer screening in gynecological outpatient clinics were subjected to liquid-based thin-layer cytology testing (TCT), high-risk human papillomavirus (hrHPV) DNA testing and PAX1/JAM3 dual-genes methylation testing (PAX1m/JAM3m). Colposcopy evaluation and biopsy were offered to women according to current guidelines. The accuracies of various testing methods and their combinations were compared based on histological diagnosis. Results: A total of 1 184 samples diagnosed by histopathology were included in this study, consisting of 541 cases (45.7%) of benign cervical tissue or chronic cervicitis, 273 (23.1%) of cervical intraepithelial neoplasia (CIN) 1, 168 (14.2%) of CIN2, 140 (11.8%) of CIN3, and 62 (5.2%) of cervical cancer. The sensitivity and specificity of PAX1m/JAM3m testing for detecting CIN2 or more severe lesions (CIN2+) were 74.1% and 95.9%, respectively. The sensitivity and specificity of PAX1m/JAM3m testing for detecting CIN3+were 87.6% and 86.8%, respectively. Receiver operating characteristic curve analysis showed that, for detecting CIN3+, the area under curve of PAX1m/JAM3m testing (0.872, 95%CI: 0.847-0.897) was significantly superior to TCT testing (0.580, 95%CI: 0.551-0.610) or hrHPV testing (0.503, 95%CI: 0.479-0.515) (all P values<0.05). Conclusions: The PAX1m/JAM3m test in cervical exfoliated cells has excellent accuracy for the diagnosis of both CIN2+and CIN3+, which is superior to traditional screening protocols and screening strategies.

Primary Ewing's sarcoma of the uterine cervix: a case report and review of the literature.

BACKGROUND: Ewing's sarcoma (ES) is an aggressive cancer of bone and soft tissue, most of which tend to occur in the bone. Extraosseous Ewing's sarcoma (EES) of the cervix is extremely rare. CASE PRESENTATION: In the present work, we reported a 39-year-old cervical EES patient with a 2.5*2.1*1.8 cm tumor mass. According to previous literatures, our case is the smallest tumor found in primary cervical ES ever. The patient initially came to our hospital due to vaginal bleeding, and then the gynecological examination found a neoplasm between the cervical canal and partially in the external cervical orifice. The diagnosis of EES was confirmed below: Hematoxylin & Eosin staining (H&E) revealed small round blue malignant cells in biopsy specimens. Immunohistochemistry (IHC) showed the positive staining for CD99, NKX2.2, and FLI1. Disruption of EWSR1 gene was found by fluorescence in situ hybridization (FISH), and the EWSR1-FLI1 gene fusion was determined by next-generation sequencing (NGS). The patient received laparoscopic wide hysterectomy, bilateral adnexectomy, pelvic lymphadenectomy, and postoperative adjuvant chemotherapy and remained disease free with regular follow-up for 1 year. CONCLUSIONS: Through a systematic review of previously reported cervical ES and this case, we highlighted the importance of FISH and NGS for the accuracy of ESS diagnosis, which could assist on the optimal treatment strategy. However, due to the rarity of the disease, there is no standard treatment schemes. Investigation on molecular pathological diagnosis and standardization of treatment regimens for cervical ES are critical to patients' prognosis.

KIF23 promotes cervical cancer progression via inhibiting NLRP3-mediated pyroptosis.

BACKGROUND: Cervical cancer (CC), closely linked to persistent human papillomavirus infection, represents a major health problem for women worldwide. The objective of this study is to elucidate KIF23's role in the development of CC and its regulatory mechanism. METHODS: The bioinformatics methods were utilized to extract pyroptosis-associated differentially expressed genes (DEGs) and pivot genes from the GSE9750 and GSE63678 datasets, followed by immune infiltration analysis and quantification of these genes' expression. The effects of kinesin family member 23 (KIF23) were verified through functional experiments in vitro and a mouse xenograft model. The NLPR3 activator, nigericin, was applied for further analyzing the potential regulatory mechanism of KIF23 in CC. RESULTS: A total of 8 pyroptosis-related DEGs were screened out, among which 4 candidate core genes were identified as candidate hub genes and confirmed upregulation in CC tissues and cells. These genes respectively showed a positive correlation with the infiltration of distinct immune cells or tumor purity. Downregulation of KIF23 could suppress the proliferation, migration, and invasion abilities in CC cells and tumorigenesis through enhancing pyroptosis. Conversely, KIF23 overexpression accelerated the malignant phenotypes of CC cells and inhibited pyroptosis activation, which was blocked by nigericin treatment. CONCLUSIONS: KIF23 may play an oncogenic role in CC progression via inhibition of the NLRP3-mediated pyroptosis pathway.

[Knockdown mitochondrial transcription factor A (TFAM) inhibits autophagy, proliferation, invasion and migration in human cervical cancer and osteosarcoma cells].

Objective To investigate the effects of mitochondrial transcription factor A (TFAM) on mitochondrial function, autophagy, proliferation, invasion, and migration in cervical cancer HeLa cells and osteosarcoma U2OS cells. Methods TFAM small-interfering RNA (si-TFAM) was transfected to HeLa and U2OS cells for downregulating TFAM expression. Mito-Tracker Red CMXRos staining combined with laser confocal microscopy was used to detect mitochondrial membrane potential (MMP). MitoSOXTM Red labeling was used to test mitochondrial reactive oxygen species (mtROS) levels. The expression of mitochondrial DNA (mtDNA) was detected by real-time quantitative PCR. Changes in the number of autophagosomes were detected by immunofluorescence cytochemistry. Western blot analysis was used to detect the expressions of TFAM, autophagy microtubule associated protein 1 light chain 3A/B (LC3A/B), autophagy associated protein 2A (ATG2A), ATG2B, ATG9A, zinc finger transcription factor Snail, matrix metalloproteinase 2 (MMP2) and MMP9. CCK-8 assay and plate clony formation assay were used to detect cell proliferation, while TranswellTM assay and scratch healing assay were used to detect changes in cell invasion and migration. Results The downregulation of TFAM expression resulted in a decrease in MMP and mtDNA copy number, but an increase in mtROS production. The protein content of LC3A/B decreased significantly compared to the control group and the number of autophagosomes in the cytoplasm decreased significantly. The expressions of ATG2B and ATG9A in the early stage of autophagy were significantly reduced. The expressions of Snail, MMP2 and MMP9 proteins in HeLa and U2OS cells were also decreased. The proliferation, invasion and migration ability of HeLa and U2OS cells were inhibited after being interfered with TFAM expression. Conclusion Downregulation of TFAM expression inhibits mitochondrial function, delays autophagy process and reduces the proliferation, invasion and migration ability of cervical cancer cells and osteosarcoma cells.