Rong Wang

Targeted demethylation of the EphA7 promoter inhibits tumorigenesis via the SP1/DNMT1 and PI3K/AKT axes and improves the response to multiple therapies in cervical cancer

Abstract Aberrant methylation of the EphA7 promoter has been observed in cervical cancer (CC); however, its precise function and role in CC remain largely unknown. In this study, we investigated the role and molecular mechanisms of EphA7 promoter methylation in cervical carcinogenesis. First, our results indicated that the reactivation of EphA7 expression via a CRISPR demethylation tool (dCas9-Tet1) had antitumor effects. It restrained tumor proliferation and invasion while promoting apoptosis via the PI3K/AKT signaling pathway in both CaSki and SiHa cells. The upstream interacting factors were subsequently captured by CRISPR-mediated pull-down in situ, and the result revealed that SP1 and MAZ interacted with the promoter of EphA7. However, the perturbation results revealed that EphA7 expression was associated with SP1/DNMT1 but not MAZ. Furthermore, 17-β-estradiol (E2) can upregulate EphA7 expression through demethylation via the SP1/DNMT1 axis. A rescue experiment revealed that interference with SP1 expression could restore the effect of E2 on increasing the expression of EphA7 by upregulating estrogen receptor expression. In addition, EphA7 demethylation reduced the half-maximal inhibitory concentration (IC50) of cisplatin and paclitaxel. Pooled analysis revealed that EphA7 promoter hypermethylation was positively correlated with tumor purity but negatively correlated with immune cell infiltration, cytotoxic T lymphocyte (CTL) and immune checkpoint (IC) activity, and the expression of EphA7 was significantly positively correlated with tumor mutational burden (TMB), microsatellite instability (MSI) and the presence of single nucleotide variant (SNV) neoantigens, suggesting a better prognosis for patients with EphA7 promoter hypomethylation and high expression. Collectively, these findings indicate that targeted demethylation of the EphA7 promoter and restoration of endogenous EphA7 expression by dCas9-Tet1 are promising therapeutic approaches and are favorable for the prognosis of CC patients.

Identification of a novel hypermethylation marker, ZSCAN18, and construction of a diagnostic model in cervical cancer

Cervical cancer (CC), a common female malignancy, has been linked to alterations in DNA methylation. This study employed an integrated "dry-wet lab" strategy combining bioinformatics, machine learning, and experimental validation to identify novel methylation biomarkers for CC. Methylome and transcriptome data from the TCGA and GEO cohorts (n=349 discovery, n=414 validation) were analyzed to identify differentially methylated CpGs. The top candidates were validated by pyrosequencing, methylation-specific PCR, and quantitative assays. Diagnostic models were developed, and functional studies were performed for the target markers. Eighteen differentially methylated CpGs were identified, with five top candidates (three in the ZSCAN18 promoter) showing diagnostic potential. ZSCAN18 promoter methylation levels and positivity rates were significantly greater in CC tissues than in normal tissues (p<0.05), reaching 77.8% (21/27) in ThinPrep cytology test (TCT) samples. The ridge regression diagnostic model achieved an AUC of 0.9421 in the validation cohort. Similarly, ZSCAN18 overexpression suppressed CC cell proliferation (p<0.05). This study established a rapid, effective and systematic systemic research strategy to screen novel methylation markers for CC. ZSCAN18 promoter methylation correlates with cervical lesion severity, and the diagnostic model enhances the diagnostic ability. These findings highlight the dual role of ZSCAN18 as a diagnostic marker and potential therapeutic target.

Reactivation of methylation‐silenced PAX1 inhibits cervical cancer proliferation and migration via the WNT/TIMELESS pathway

AbstractAlthough aberrant methylation of PAX1 is closely associated with cervical cancer (CC), PAX1 methylation (PAX1m) and its role in CC remain to be elucidated. Here, we clarified the biological function of PAX1 in CC. First, PAX1m in ThinPrep cytologic test samples was measured via quantitative methylation‐specific PCR. The results showed that PAX1 promoter methylation levels were significantly increased in CC patients (p &lt; 0.001). We also found that PAX1 promoter methylation levels were positively correlated with tumor purity but negatively correlated with immune‐infiltration via public databases. Then, CRISPR‐based methylation perturbation tools (dCas9‐Tet1) were constructed to further demonstrate that DNA methylation participates in the regulation of PAX1 expression directly. Gain‐ and loss‐of‐function experiments were used to show that PAX1 overexpression restrained proliferation, migration and improved cisplatin sensitivity by interfering with the WNT/TIMELESS axis in CC cells. Additionally, Co‐immunoprecipitation assays further confirmed the interaction between PAX1 and TCF7L2. Taken together, our results suggested that a tumor suppressor role of PAX1 in CC and that CRISPR‐based PAX1 demethylation editing might be a promising therapeutic strategy for CC.

SOX14 hypermethylation as a tumour biomarker in cervical cancer

AbstractBackgroundThe association between SOX14 and cancer has been reported. The aim of this study was to identify and validate the potential value of SOX14 methylation in the early detection of cervical cancer.MethodsFirst, we extracted the data for SOX14 methylation and expression within cervical cancer from The Cancer Genome Atlas (TCGA) database and analysed them via UALCAN, Wanderer, MEXPRESS and LinkedOmics. Subsequently, according to the bioinformatics findings, primers and probes were designed for the most significantly differentiated methylation CpG site and synthesized for methylation-specific PCR (MSP) and quantitative methylation-specific PCR (QMSP) to verify SOX14 methylation in both cervical tissuses and liquid-based cell samples. Eventually, the clinical diagnostic efficacy of SOX14 methylation in the normal, cervical intraepithelial neoplasia, and cancer groups was analysed by ROCAUC.ResultsPooled analysis demonstrated that SOX14 methylation levels were significantly increased in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) compared to normal tissues (P &lt; 0.001). Both the verification and validation cohorts indicated that the methylation level and the positive rate of SOX14 gradually increased with increasing severity from normal to cancer samples (P &lt; 0.01). When the cut-off value was set as 128.45, the sensitivity and specificity of SOX14 hypermethylation in the diagnosis of cervical cancer were 94.12 and 86.46%, respectively. When taken as a screening biomarker (&gt;CINII), the sensitivity was 74.42% and the specificity was 81.48%, with a cut-off value of 10.37.ConclusionSOX14 hypermethylation is associated with cervical cancer and has the potential to be a molecular biomarker for the screening and early diagnosis of cervical cancer.

Chemotherapy versus chemoradiotherapy for FIGO stages IB1 and IIA1 cervical squamous cancer patients with lymphovascular space invasion: a retrospective study

Abstract Purpose To evaluate the impact of different adjuvant therapy on IB1 and IIA1 stage cervical squamous cell cancer patients with lymphovascular space invasion. It also aimed to analyze the relationship between lymphovascular space invasion and other clinical pathological characteristics on IB1 and IIA1 stage cervical squamous cell cancer patients. Methods This retrospective observational study collected data of FIGO stages IB1 and IIA1 squamous cervical cancer patients at the First Affiliated Hospital of Chongqing Medical University between 2014 and 2018. A correlation analysis between lymphovascular space invasion and other clinical or pathological factors was conducted. Prognosis analysis of patients with lymphovascular space invasion were performed to assess associations between clinical-pathological characteristics and survival. Results A total of 357 women were identified including 110 (30.8%) with lymphovascular space invasion, 247 (69.2%) without lymphovascular space invasion. Both middle 1/3 cervical stromal invasion (p = 0.000) and deep 1/3 cervical stromal invasion (p = 0.000) were independently associated with lymphovascular space invasion. Among lymphovascular space involved women, tumor differentiation (P = 0.001) and postoperative therapy (P = 0.036) had a significant influence on disease recurrence. Multivariate analysis showed that lymph node metastasis (P = 0.017), poorer tumor differentiation (P = 0.036) and postoperative chemotherapy alone (P = 0.021) can increase the risk of tumor relapse. Conclusion Our study suggested that the presence of deep stromal invasion independently increases the risk of lymphovascular space invasion. Compared with chemotherapy, chemoradiotherapy seems to improve progression-free survival in squamous cervical cancer patients with lymphovascular space invasion.

Assessing the influence of gestational trophoblastic disease history on IVF-ET pregnancy outcomes: a retrospective cohort study

To evaluate the impact of a history of gestational trophoblastic disease on pregnancy outcomes in in vitro fertilization-embryo transfer procedures(IVF-ET) and to utilize logistic regression to analyze potential risk factors influencing re-pregnancy outcomes among women with GTD histories undergoing IVF-ET. This retrospective cohort study collected data from patients with a history of GTD who underwent IVF-ET at the hospital from January 2018 to January 2023. The study group comprised 27 women with a history of GTD. A control group of 54 women, matched at a 1:2 ratio, without a GTD history, was selected from those who underwent IVF-ET during the same period at the same hospital. Statistical analyses were employed to compare baseline characteristics, embryological parameters, and pregnancy outcomes between the two groups. The study group exhibited significantly lower endometrial thickness (EMT) during the mid-luteal phase of the natural menstrual cycle, reduced EMT on the day of embryo transfer (ET), and a decreased blastocyst formation rate compared to the control group (p < 0.05). Furthermore, the study group had a higher number of uterine curettages prior to transfer, an increased rate of discarded embryos, and higher total sperm motility (PR + NP) in their spouses (p < 0.05). Logistic regression analysis revealed that a history of GTD does not significantly affect pregnancy outcomes post-IVF-ET. A history of GTD does not significantly influence pregnancy outcomes following IVF-ET. Therefore, patients with a GTD history and their physicians can approach the IVF-ET process with less anxiety and adopt a more positive and rational outlook.