PAX1 hypomethylation as a prognostic biomarker for radioresistance of cervical cancer.

BACKGROUND: PAX1 gene methylation plays an important role in the development of cervical cancer. However, its prognostic value after radiotherapy for locally advanced cervical cancer is unknown, so this study aimed to investigate the value of PAX1 gene methylation for predicting the sensitivity of radiotherapy for cervical cancer. METHODS: We selected 125 patients with primary cervical cancer who underwent concurrent chemo-radiotherapy as the study population, quantitative methylation-specific polymerase chain reaction (QMSP) was used for detecting PAX1 methylation status of cervical exfoliated cells. Logistic regression model was used to analyze the risk factors associated with the short-term efficacy and to establish a prediction model of radiotherapy sensitivity based on PAX1 gene methylation. Cell viability after radiation of Hela and SiHa cells transfected with PAX1 or control vector was evaluated by CCK8. Furthermore, RNA-Seq analyses identified different expressed genes (DEGs) in PAX1 overexpressed SiHa cells. Gene Ontology (GO) and pathway enrichment analysis was carried out to determine the biological function of DEGs. RESULTS: PAX1 methylation level was associated with HPV16/18-positive rate. PAX1 hypomethylation was found to be a risk factor for tumor residual after chemo-radiotherapy. A nomogram containing the risk factors for PAX1 methylation status, lymph node metastasis, pathological type and tumor size was further constructed to predict the probability of tumor residual after chemo-radiotherapy (AUC = 0.823, 95% CI 0.736-0.910). High PAX1 protein level was more likely to cause radioresistance in both Hela and SiHa cells. Transcriptomic sequencing of PAX1 overexpressed and control cells identified 615 differentially expressed genes, and GO enrichment analysis suggested that PAX1 may be involved in the regulation of signaling receptor activity and response to viruses. CONCLUSION: PAX1 hypomethylation status could be used as a promising biomarker to predict radioresistance in cervical cancer. This further provides a new idea for the individualized treatment strategy of simultaneous radiotherapy for cervical cancer.

CCNE1 is a potential target of Metformin for tumor suppression of ovarian high-grade serous carcinoma.

High-grade serous ovarian cancer (HGSOC) is the most common and malignant type of ovarian cancer, accounting for 70%-80% of mortality. However, the treatment of HGSOC has improved little in the past few decades. Metformin is the first-line medication for the treatment of type 2 diabetes and has now gained more attention in cancer treatment. In this study, we sought to identify potential hub genes that metformin could target in the treatment of HGSOC. We downloaded GSE69428 and GSE69429 in the Gene Expression Omnibus database and performed the bioinformatics analysis. Subsequently, we analyzed the effect of Metformin in HGSOC through biological experiments. Molecular simulation docking was used to predict the interaction of Metformin and CCNE1. We chose CCNE1 for the study based on bioinformatics analysis, literature studies, and preliminary data. We evaluated that CCNE1 is overexpressed in HGSOC tissues and found that HGSOC cells with high CCNE1 expression increase sensitivity to Metformin treatment in the analysis of cell proliferation and anchorage-independent growth. Metformin could inhibit the expression of CCNE1, which is associated with the anti-proliferative effect of tumor cells. Moreover, Metformin could ameliorate the tumor growth in syngeneic orthotopic transplantation mouse models and xenograft tumorigenesis models. Furthermore, molecular simulation docking showed that Metformin may bind to CCNE1 protein, suggesting that CCNE1 could be a potential target for Metformin. Our data revealed that Metformin has antitumor effects on ovarian cancer and CCNE1 could be a potential target for Metformin.

Gut microbiota and host Cyp450s co-contribute to pharmacokinetic variability in mice with non-alcoholic steatohepatitis: Effects vary from drug to drug.

INTRODUCTION: Pharmacokinetic variability in disease state is common in clinical practice, but its underlying mechanism remains unclear. Recently, gut microbiota has been considered to be pharmacokinetically equivalent to the host liver. Although some studies have explored the roles of gut microbiota and host Cyp450s in drug pharmacokinetics, few have explored their effects on pharmacokinetic variability, especially in disease states. OBJECTIVES: In this study, we aim to investigate the effects of gut microbiota and host Cyp450s on pharmacokinetic variability in mice with non-alcoholic steatohepatitis (NASH), and to elucidate the contribution of gut microbiota and host Cyp450s to pharmacokinetic variability in this setting. METHODS: The pharmacokinetic variability of mice with NASH was explored under intragastric and intravenous administrations of a cocktail mixture of omeprazole, phenacetin, midazolam, tolbutamide, chlorzoxazone, and metoprolol, after which the results were compared with those obtained from the control group. Thereafter, the pharmacokinetic variabilities of all drugs and their relations to the changes in gut microbiota and host Cyp450s were compared and analyzed. RESULTS: The exposures of all drugs, except metoprolol, significantly increased in the NASH group under intragastric administration. However, no significant increase in the exposure of all drugs, except tolbutamide, was observed in the NASH group under intravenous administration. The pharmacokinetic variabilities of phenacetin, midazolam, omeprazole, and chlorzoxazone were mainly associated with decreased elimination activity in the gut microbiota. By contrast, the pharmacokinetic variability of tolbutamide was mainly related to the change in the host Cyp2c65. Notably, gut microbiota and host Cyp450s exerted minimal effects on the pharmacokinetic variability of metoprolol. CONCLUSION: Gut microbiota and host Cyp450s co-contribute to the pharmacokinetic variability in mice with NASH, and the degree of contribution varies from drug to drug. The present findings provide new insights into the explanation of pharmacokinetic variability in disease states.

Effect of Washed Microbiota Transplantation on Patients With Dyslipidemia in South China.

Background and Aims: Although the manual crude fecal microbiota transplantation (FMT) reduces blood lipids in animal models of hyperlipidemia, its clinical effect on blood lipid metabolism in patients with hyperlipidemia and hypolipidemia remains unclear, especially in the Chinese population. It was reported that washed microbiota transplantation (WMT) was safer, more precise, and more quality-controllable than the crude FMT by manual. This study aimed to investigate the feasibility and effectiveness of WMT on lipid metabolism in the Chinese population. Methods: Clinical data of patients with various indications who received WMT for 1-3 treatment procedures were collected. Changes in blood lipids before and after WMT, namely, total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), homeostasis model assessment of insulin resistance (HOMA-IR), liver fat attenuation, and liver stiffness measurement, were compared. Results: A total of 177 patients (40 cases of hyperlipidemia, 87 cases with normal blood lipids, and 50 cases of hypolipidemia) were enrolled in the First Affiliated Hospital of Guangdong Pharmaceutical University. WMT has a significant therapeutic effect in reducing blood lipid levels (TC and TG) in the short- and medium term in patients with hyperlipidemia (p <0.05). Hyper blood lipid decreased to normal in the short-term (35.14%; p <0.001), and LDL-C changed to normal in the medium term (33.33%; p = 0.013). In the hypolipidemia group, 36.36% and 47.06% changed to normal in the short-term (p = 0.006) and medium term (p = 0.005) of therapeutic effects based on blood lipid levels. In the normal blood lipid group and the low-risk group of atherosclerotic cardiovascular disease (ASCVD), the change was not statistically significant, indicating that WMT does not increase the risk of blood lipid and ASCVD in the long-term. Conclusions: WMT treatment changes blood lipids in patients with hyperlipidemia and hypolipidemia without serious adverse events, with no risk for increasing blood lipids and ASCVD in the long-term. There were significant decreased TC, TG, and LDL-C levels in the medium term of WMT treatment for hyperlipidemia. Therefore, the regulation of gut microbiota by WMT may indicate a new clinical method for the treatment of dyslipidemia.

A Joint Technology Combining the Advantages of Capillary Microsampling with Mass Spectrometry Applied to the Trans-Resveratrol Pharmacokinetic Study in Mice.

Mice are the most frequently used animals in pharmacokinetic studies; however, collecting series of blood samples from mice is difficult because of their small sizes and tiny vessels. In addition, due to the small sample size, it is problematic to perform high required quantification. Thus, present work aims to find an effective strategy for overcoming these challenges using trans-resveratrol as a tool drug. Based on the idea of a joint technology, the capillary microsampling (CMS) was chosen for blood sample collection from mice after delivery of trans-resveratrol (150 mg/kg) by gavage, and a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of trans-resveratrol and its main metabolites. All the mouse blood samples were exactly collected by CMS without obvious deviation. This provided credible samples for subsequent quantitative analysis. The HPLC-MS/MS method was found to be sensitive, accurate, and repeatable, and the pharmacokinetic parameters for all analytes were comparable with those reported in previous studies. However, the present joint technology offers the advantages of less animal damage, easy for sample preparation, and improved reliability. It has overcome some of the major limitations revealed in previous pharmacokinetic studies in mice and therefore provides a more effective option for future studies.

Imbalance of the Gut Microbiota May Be Associated with Missed Abortions: A Perspective Study from a General Hospital of Hunan Province.

OBJECTIVE: To conduct a preliminary investigation that shows the possible correlation between the change of gut microbiota and missed abortions (MAs), which further provides a new potential insight for the prevention and therapy of MAs. METHOD: One hundred women, including 50 patients with MAs (case group) and 50 normal pregnant women (control group), were enrolled in the study. Fecal specimens were collected in the first trimester. Bacterial DNA was extracted, hybridized with primers of specific genes, and then detected by bacterial chip. The composition and the relative abundance of the gut microbiota were compared and analyzed. Furthermore, Kyoto Encyclopedia of Genes and Genomes enrichment analysis was used to explore the relative pathways. RESULTS: (1) The α-diversity and ß-diversity of the gut microbiota in patients with MAs were significantly lower than that those in normal pregnant women (P < 0.05). At the phylum level, Firmicutes, Proteobacteria, Actinomycetes, and Bacteroidetes accounted for the main proportion of intestinal flora in the 2 groups. Only Actinobacteria was high in the case group. Significant differences were found between the two groups at the phylum level (P < 0.05). Prevotella, Lactobacillus, and Paracoccus were significantly more abundant in the control group than in the case group at the genus level (P < 0.05). (2) KEGG pathway enrichment analysis found significant differences in 27 signaling pathways and metabolic pathways between the two groups of differentially expressed genes (all adjusted P < 0.05). (3) The positive rate of M. hominins (MH) detection in the control group was significantly higher in the MA group (χ 2 = 7.853, P = 0.004). CONCLUSION: The high abundance of Actinobacteria in the MA group was the first time found and reported in the study. The dysbiosis of the gut microbiota correlates with MAs. This study provided insights into the potential change of gut microbiota of MAs and the potential underlying mechanisms through certain impaired lipid metabolism and aroused inflammation pathways. Comprehensive insights regarding gut microbiota may facilitate improved understanding and the development of novel therapeutic and preventive strategies for MAs.

Washed Microbiota Transplantation Lowers Blood Pressure in Patients With Hypertension.

Background: Although transplantation of the fecal microbiota from normotensive donors has been shown to have an antihypertensive effect in hypertensive animal models, its effect on blood pressure in patients with hypertension is unclear. This study aimed to assess the effect of washed microbiota transplantation (WMT) from normotensive donors on blood pressure regulation in hypertensive patients. Methods: The clinical data of consecutive patients treated with washed microbiota transplantation (WMT) were collected retrospectively. The blood pressures of hypertensive patients before and after WMT were compared. The factors influencing the antihypertensive effect of WMT in hypertensive patients and fecal microbial composition of donors and hypertensive patients were also analyzed. Results: WMT exhibited an antihypertensive effect on blood pressure: the blood pressure at hospital discharge was significantly lower than that at hospital admission (change in systolic blood pressure: -5.09 ± 15.51, P = 0.009; change in diastolic blood pressure: -7.74 ± 10.42, P < 0.001). Hypertensive patients who underwent WMT via the lower gastrointestinal tract (ß = -8.308, standard error = 3.856, P = 0.036) and those not taking antihypertensive drugs (ß = -8.969, standard error = 4.256, P = 0.040) had a greater decrease in systolic blood pressure, and hypertensive patients not taking antihypertensive drugs also had a greater decrease in diastolic blood pressure (ß = -8.637, standard error = 2.861, P = 0.004). After WMT, the Shannon Diversity Index was higher in six of eight hypertensive patients and the microbial composition of post-WMT samples tended to be closer to that of donor samples. Conclusion: WMT had a blood pressure-lowering effect in hypertensive patients, especially in those who underwent WMT via the lower gastrointestinal tract and in those not taking antihypertensive drugs. Therefore, modulation of the gut microbiota by WMT may offer a novel approach for hypertension treatment.

Methylation of PAX1 gene promoter in the prediction of concurrent chemo-radiotherapy efficacy in cervical cancer.

Objectives: Cervical cancer is the fourth leading cause of cancer death among women worldwide. In currently, aberrant methylation of PAX1 is found in variety of solid tumors, including cervical cancer. In addition, the role of PAX1 gene methylation in cervical cancer and precancerous lesions screening has been confirmed in previous study. Here, we evaluated the predictive value of PAX1 methylation in concurrent chemo-radiotherapy (CCRT) outcomes in cervical cancer. Methods: This study enrolled 82 cervical cancer patients from August 2018 to August 2020. We compared the clinical results between different PAX1 methylation status. Hyper-methylation patients were subjects to MRI and quantitative methylation-specific PCR (QMSP) for PAX1 before, in the middle, immediately after, 1 month and 3 months after CCRT. The changes in PAX1 methylation during CCRT were analyzed. Results: The lower PAX1 methylation status were related to a poor tumor response. Based on the MRI findings three months post-treatment, the hypermethylated patients were classified into the complete response (CR; n=50) and partial remission (PR; n=18) groups. The average PAX1 △Cp value of CR and PR groups before radiotherapy was 5.08±1.98 and 4.32±2.00 respectively, and after concurrent chemo-radiotherapy was significantly increased to 17.35±4.96 and 16.99±6.17, respectively (P<0.05). Furthermore, the PAX1 △Cp value between CR and PR groups were significantly different at mid-treatment and performed well in predicting short-term efficacy (AUC 0.84) in this period, and its sensitivity and specificity for predicting PR were 0.72 and 0.88, respectively. Conclusion: The PAX1 methylation level may predict the sensitivity and efficacy of CCRT in cervical cancer.

The characteristics and risk factors of human papillomavirus infection: an outpatient population-based study in Changsha, Hunan.

This cross-sectional study investigated the characteristics of cervical HPV infection in Changsha area and explored the influence of Candida vaginitis on this infection. From 11 August 2017 to 11 September 2018, 12,628 outpatient participants ranged from 19 to 84 years old were enrolled and analyzed. HPV DNA was amplified and tested by HPV GenoArray Test Kit. The vaginal ecology was detected by microscopic and biochemistry examinations. The diagnosis of Candida vaginitis was based on microscopic examination (spores, and/or hypha) and biochemical testing (galactosidase) for vaginal discharge by experts. Statistical analyses were performed using SAS 9.4. Continuous and categorical variables were analyzed by t-tests and by Chi-square tests, respectively. HPV infection risk factors were analyzed using multivariate logistic regression. Of the total number of participants, 1753 were infected with HPV (13.88%). Females aged ≥ 40 to < 50 years constituted the largest population of HPV-infected females (31.26%). The top 5 HPV subtypes affecting this population of 1753 infected females were the following: HPV-52 (28.01%), HPV-58 (14.83%), CP8304 (11.47%), HPV-53 (10.84%), and HPV-39 (9.64%). Age (OR 1.01; 95% CI 1-1.01; P < 0.05) and alcohol consumption (OR 1.30; 95% CI 1.09-1.56; P < 0.01) were found to be risk factors for HPV infection. However, the presence of Candida in the vaginal flora was found to be a protective factor against HPV infection (OR 0.62; 95% CI 0.48-0.8; P < 0.001). Comparing with our previous study of 2016, we conclude that the subtype distribution of HPV infection is relatively constant in Changsha. Our data suggest a negative correlation between vaginal Candida and HPV, however, more radical HPV management is required in this area for perimenopausal women and those who regularly consume alcohol.

TruScreen detection of cervical tissues for high-risk human papillomavirus-infected women during the COVID-19 pandemic.

Aims: To evaluate the efficacy of TruScreen (TS01) for high-risk human papillomavirus (HR-HPV) women compared with other methods in reducing colposcopy referral rates in hospitals. Methods: A single-center, prospective, case-control study was conducted from December 2019 to June 2020. Results: Among 139 (46.2%) HR-HPV-positive patients, 58 were CIN1, 52 were CIN2-3 and 29 had cervical cancer (n = 29). The sensitivity and specificity of detecting CIN2+ by TS01, colposcopy and HPV16/18 testing were 96.3% and 46.4%, 85.2% and 40.5% and 59.3% and 74.1%, respectively. The highest sensitivity was 96.3% at HPV16/18 and TS01 (each positive results), and the highest specificity was 83.6% at HPV16/18 and TS01 (both positive) for CIN2+ compared with the other methods. Conclusion: TS01 is a noninvasive screening method and can be used to diagnose cervical lesions quickly. It is especially suitable as triage tool for HR-HPV-positive women facing SARS-CoV-2 exposure and infection risks in hospital.

CSF3 Is a Potential Drug Target for the Treatment of COVID-19.

Coronavirus Disease 2019 (COVID-19) is an acute respiratory infectious disease that appeared at the end of 2019. As of July 2020, the cumulative number of infections and deaths have exceeded 15 million and 630,000, respectively. And new cases are increasing. There are still many difficulties surrounding research on the mechanism and development of therapeutic vaccines. It is urgent to explore the pathogenic mechanism of viruses to help prevent and treat COVID-19. In our study, we downloaded two datasets related to COVID-19 (GSE150819 and GSE147507). By analyzing the high-throughput expression matrix of uninfected human bronchial organoids and infected human bronchial organoids in the GSE150819, 456 differentially expressed genes (DEGs) were identified, which were mainly enriched in the cytokine-cytokine receptor interaction pathway and so on. We also constructed the protein-protein interaction (PPI) network of DEGs to identify the hub genes. Then we analyzed GSE147507, which contained lung adenocarcinoma cell lines (A549 and Calu3) and the primary bronchial epithelial cell line (NHBE), obtaining 799, 460, and 46 DEGs, respectively. The results showed that in human bronchial organoids, A549, Calu3, and NHBE samples infected with SARS-CoV-2, only one upregulated gene CSF3 was identified. Interestingly, CSF3 is one of the hub genes we previously screened in GSE150819, suggesting that CSF3 may be a potential drug target. Further, we screened potential drugs targeting CSF3 by MOE; the top 50 drugs were screened by flexible docking and rigid docking, with 37 intersections. Two antiviral drugs (Elbasvir and Ritonavir) were included; Elbasvir and Ritonavir formed van der Waals (VDW) interactions with surrounding residues to bind with CSF3, and Elbasvir and Ritonavir significantly inhibited CSF3 protein expression.

Aberrant DNA methylation of PAX1, SOX1 and ZNF582 genes as potential biomarkers for esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a highly invasive malignant tumor and the majority of patients have advanced stage of ESCC at diagnosis with poor outcome. Identification of sensitive and specific biomarkers for early screening of ESCC is critical for improving patient overall survival. METHODS: Pyrosequencing was used to determine the magnitude of DNA methylation on the selected regions PAX1 (paired box gene1), SOX1(sex determining region Y-box-1), and ZNF582 (zinc finger protein 582) in ESCC. RESULTS: The methylation levels ofPAX1, SOX1, and ZNF582 genes were significantly higher in the cancerous tissues compared to those in the non-cancerous (all P < 0.0001). The accuracy, sensitivity and specificity of PAX1 methylation for the detection of cancer were respectively 0.754, 96.0% and 51.4%; for SOX1 which were 0.781, 89.2% and 59.5%; for ZNF582 which were 0.898, 93.2% and 75.7%. Most importantly, both the sensitivity and specificity of ZNF582 methylation testing achieved 100% in female ESCC patients. Hypermethylation of PAX1/SOX1/ZNF582 exhibited as an independent risk factor for ESCC development. In addition, ZNF582 methylation level in tumor tissue from the female patients was higher than that from male patients, and it was higher in the moderate and poor differentiated tumors compared to that in well-differentiated tumors. SOX1 methylation level was significantly higher in tumors located in the upper region than those located in the middle or lower regions. CONCLUSION: The methylation levels ofPAX1, SOX1 and ZNF582 genes were all higher in cancer tissues than in paired-adjacent and normal tissues, suggesting that detection of PAX1/SOX1/ZNF582 methylation status may serve as a promising biomarker for ESCC screening and diagnosis.

High methylation of ZNF582 in cervical adenocarcinoma affects radiosensitivity and prognosis.

BACKGROUND: Our previous study demonstrated hypermethylation of the ZNF582 gene in cervical cancer, but its prognostic value in cervical cancer, especially in cervical adenocarcinoma (CAC), remains unclear. The present study aimed to investigate the value of ZNF582 gene methylation for diagnosis and prediction of radiochemotherapy sensitivity and prognosis in CAC. METHODS: We first determined ZNF582 methylation levels using quantitative methylation-specific PCR in a training set. Disease-free survival and overall survival (DFS and OS) rates were estimated using the Kaplan-Meier method. A Cox regression model was used to assess the prognostic significance of ZNF582 gene methylation in CAC patients. Immunohistochemistry was used to test ZNF582 protein expression in CAC tissues, and an MTT assay evaluated the sensitivity of Hela cells (with or without ZNF582 transfection) to radiation and chemotherapy. RESULTS: The ZNF582 gene showed a higher level of methylation in the CAC group than in the noncancer group, and patients negative for ZNF582 methylation had worse prognoses. We also found that ZNF582 methylation levels were reduced in concomitant chemo-radio-therapy (NCRT) patients compared with that in non-NCRT patients. Methylation-negative status was correlated with high ZNF582 protein expression, and ZNF582 protein overexpression could increase resistance to radiation and chemotherapy in Hela cells. CONCLUSIONS: Aberrant high methylation of ZNF582 may be a potential biomarker for CAC detection and prognosis monitoring. Overexpression of ZNF582 protein could increase CAC chemoradiotherapy resistance.

The promising role of PAX1 (aliases: HUP48, OFC2) gene methylation in cancer screening.

BACKGROUND: Paired-box gene 1 (PAX1), a member of the PAX family, plays a role in pattern formation during embryogenesis, and might be essential for development of the vertebral column. METHODS: PAX1 is silenced by methylation in several cancers and is considered a tumor suppressor gene. Our previous studies reported PAX1 as hypermethylated in cervical cancer tissues, thereby suggesting it as a potential screening marker. Recently, an increasing number of studies have confirmed PAX1 methylation as a promising biomarker in cervical cancer based on its excellent discriminatory ability between high-grade cervical lesions and normal tissues, resulting in a reduced necessity for referral for colposcopy and biopsy. Additionally, PAX1 is also hypermethylated in other tumors, including those associated with epithelial ovarian cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, and endometrial carcinoma, and shows relatively good sensitivity and specificity for the detection of these tumors. RESULTS: This review summarizes reports of PAX1 methylation and its promising role in cancer screening, especially that associated with cervical cancer. CONCLUSION: According to current evidence, combined testing for human papillomavirus and PAX1 methylation analysis represents an efficacious cervical cancer-screening protocol.

Neoadjuvant chemotherapy for locally invasive cervical cancer in pregnancy: two case reports.

Although cervical cancer is the most common gynecological cancer, it is rare in pregnant women. Treatment of locally invasive cervical cancer during pregnancy is often complex and challenging. Neoadjuvant chemotherapy (NACT) is a possible treatment option. Here we report two cases of cervical cancer diagnosed during pregnancy, one of which was sensitive to NACT, while the other was not sensitive to chemotherapy but showed a good response to concurrent chemoradiotherapy (CCRT) and intra-arterial chemotherapy. Both of these cases have not revealed any signs of tumor recurrence and their children are growing healthily.

Utility of gene methylation analysis, cytological examination, and HPV-16/18 genotyping in triage of high-risk human papilloma virus-positive women.

In 2015, the American Society for Colposcopy and Cervical Pathology and the Society of Gynecologic Oncology issued interim guidance for the use of a human papillomavirus (HPV) test for primary screening, suggesting triage of women positive for high-risk human papillomavirus (hrHPV) by HPV-16/18 genotyping and cytology for women positive for non-16/18 hrHPV. The design of the present study was based on this interim guidance and analysis of the methylation status of specific candidate genes, which has been proposed as a tool to reduce unnecessary referral following primary HPV screening for cervical cancer. We performed a hospital-based case-control study including 312 hrHPV-positive women. hrHPV genotyping was performed by nested multiplex PCR assay with type-specific primers.Residual cervical cells from liquid-based cytology were used for extraction of genomic DNA for assessment of the methylation status of PAX1, ZNF582, SOX1, and NKX6-1 and HPV genotyping. Combined with HPV-16/18 genotyping, both a dual methylation test for PAX1/ZNF582 and testing for ZNF582 methylation demonstrated 100% association of methylation with pathology results, indicating carcinoma in situ or squamous cell carcinoma. The sensitivity and specificity of the dual methylation test for PAX1/ZNF582 as a reflex test for identification of CIN3+ lesions were 78.85% and 73.55% (odds ratio = 10.37, 95% confidence interval = 4.76-22.58), respectively. This strategy could reduce the number of patients referred for colposcopic examination by 31.3% compared with cytology, and thus provide a feasible follow-up solution in regions where colposcopy is not readily available. This strategy could also prevent unnecessary anxiety in women with hrHPV infection.

DNA Methylation Status of PAX1 and ZNF582 in Esophageal Squamous Cell Carcinoma.

Hypermethylation of specific gene promoters is an important mechanism of carcinogenesis. A high frequency of promoter methylation of PAX1 and ZNF582 genes has been detected in cervical cancer. In the present study, we investigated the methylation status of PAX1 and ZNF582 genes in esophageal squamous cell carcinoma (ESCC) tissues. Tumor and paracancerous tissues were obtained from 14 ESCC patients. Genomic DNA was extracted from both tumor and paracancerous tissues, and the concentration of DNA were determined. DNA methylation analysis of PAX1 and ZNF582 genes was carried out using quantitative methylation-specific PCR. To assess the diagnostic performance of the two methylated genes for cancer detection, receiver operating characteristic (ROC) curves were generated. Sensitivities and specificities were tested at cut-offs obtained from the ROC curves. The methylation levels of both PAX1 and ZNF582 genes were significantly higher in tumor tissues compared to non-tumor paracancerous tissues. The methylation rates of PAX1 and ZNF582 in ESCC tumor and paracancerous tissues were 100% and 21.4% (p = 0.006), 85.7% and 0% (p < 0.001), respectively. The sensitivities and specificities of PAX1 and ZNF582 methylation for the detection of cancer were 100% and 85.7%, and 78.6% and 100%, respectively. The DNA methylation levels and frequencies of PAX1 and ZNF582 genes were markedly higher in ESCC tumor tissues compared to those in paracancerous tissues. Moreover, the conclusions were verified by using The Cancer Genome Atlas (TCGA) datasets. DNA methylation status of these two genes showed a relatively good sensitivity and specificity for the detection of ESCC tumors. This data suggests that DNA methylation testing holds a great promise for ESCC screening and warrants further prospective population-based studies.

Hypermethylated ZNF582 and PAX1 are effective biomarkers for detection of oral dysplasia and oral cancer.

OBJECTIVES: This study investigated whether the methylation of ZNF582, PAX1, SOX1, NKX6.1, and PTPRR genes in oral scrapings could be used to detect oral dysplasia and oral cancer and to predict oral cancer recurrence. MATERIALS AND METHODS: Oral scrapings were collected from 65 normal oral mucosa subjects, 107 oral precancer patients, and 95 oral squamous cell carcinoma patients. Methylation levels of the five genes were quantified by real-time methylation-specific PCR after bisulfite conversion. RESULTS: Among the five tested genes, methylated ZNF582 (ZNF582m) and PAX1 (PAX1m) were found to be appropriate biomarkers for oral dysplasia and oral cancers. ZNF582m could detect mild dysplasia or worse oral lesions with the sensitivity and specificity being 0.85 and 0.87, respectively. PAX1m performed better in identifying moderate dysplasia or worse oral lesions with the sensitivity and specificity being 0.72 and 0.86, respectively. Moreover, the methylation levels and positive rates for ZNF582m and PAX1m were increased when disease severity increased. Thus, they may be applicable as a triage tool for patients with abnormal visual oral examinations. After cancer excision, both ZNF582m and PAX1m levels decreased. However, their levels increased again at the subsequently recurrent sites in some patients approximately 3-4 months before cancer recurrence. Finally, areca-quid chewing alone and in combination with cigarette smoking or alcohol drinking were found to be correlated with ZNF582 and PAX1 hypermethylation. CONCLUSION: We conclude that hypermethylated ZNF582 and PAX1 are effective biomarkers for the detection of oral dysplasia and oral cancer and for the prediction of oral cancer recurrence.

Real-time colorimetric detection of DNA methylation of the PAX1 gene in cervical scrapings for cervical cancer screening with thiol-labeled PCR primers and gold nanoparticles.

BACKGROUND: DNA methylation can induce carcinogenesis by silencing key tumor suppressor genes. Analysis of aberrant methylation of tumor suppressor genes can be used as a prognostic and predictive biomarker for cancer. In this study, we propose a colorimetric method for the detection of DNA methylation of the paired box gene 1 (PAX1) gene in cervical scrapings obtained from 42 patients who underwent cervical colposcopic biopsy. METHODS: A thiolated methylation-specific polymerase chain reaction (MSP) primer was used to generate MSP products labeled with the thiol group at one end. After bisulfite conversion and MSP amplification, the unmodified gold nanoparticles (AuNPs) were placed in a reaction tube and NaCl was added to induce aggregation of bare AuNPs without generating polymerase chain reaction products. After salt addition, the color of AuNPs remained red in the methylated PAX1 gene samples because of binding to the MSP-amplified products. By contrast, the color of the AuNP colloid solution changed from red to blue in the non-methylated PAX1 gene samples because of aggregation of AuNPs in the absence of the MSP-amplified products. Furthermore, PAX1 methylation was quantitatively detected in cervical scrapings of patients with varied pathological degrees of cervical cancer. Conventional quantitative MSP (qMSP) was also performed for comparison. RESULTS: The two methods showed a significant correlation of the methylation frequency of the PAX1 gene in cervical scrapings with severity of cervical cancer (n=42, P<0.05). The results of the proposed method showed that the areas under the receiver operating characteristic curve (AUCs) of PAX1 were 0.833, 0.742, and 0.739 for the detection of cervical intraepithelial neoplasms grade 2 and worse lesions (CIN2+), cervical intraepithelial neoplasms grade 3 and worse lesions (CIN3+), and squamous cell carcinoma, respectively. The sensitivity and specificity for detecting CIN2+ lesions were 0.941 and 0.600, respectively, with a cutoff value of 31.27%. The proposed method also showed superior sensitivity over qMSP methods for the detection of CIN2+ and CIN3+ (0.941 vs 0.824 and 1.000 vs 0.800, respectively). Furthermore, the novel method exhibited higher AUC (0.833) for the detection of CIN2+ than qMSP (0.807). CONCLUSION: The results of thiol-labeled AuNP method were clearly observed by the naked eyes without requiring any expensive equipment. Therefore, the thiol-labeled AuNP method could be a simple but efficient strategy for cervical cancer screening.

Combined clinical and genetic testing algorithm for cervical cancer diagnosis.

BACKGROUND: Opportunistic screening in hospitals is widely used to effectively reduce the incidence rate of cervical cancer in China and other developing countries. This study aimed to identify clinical risk factor algorithms that combine gynecologic examination and molecular testing (paired box gene 1 (PAX1) or zinc finger protein 582 (ZNF582) methylation or HPV16/18) results to improve diagnostic accuracy. METHODS: The delta Cp of methylated PAX1 and ZNF582 was obtained via quantitative methylation-specific PCR in a training set (57 CIN2- and 43 cervical intraepithelial neoplasia ≥grade 3 (CIN3+) women), and the individual and combination gene sensitivities and specificities were determined. The detection accuracy of three algorithms combining gynecologic findings and genetic test results was then compared in a randomized case-control study comprising 449 women referred for colposcopic examination by gynecologists in the outpatient department of Xiangya Hospital between November 2011 and March 2013. RESULTS: Significant association was observed between CIN3+ and methylated PAX1 or ZNF582 in combination with HPV16/18 (OR:15.52, 95 % CI:7.73-31.18). The sensitivities and specificities of methylated PAX1 or ZNF582 combined with HPV16/18 for CIN3+ women were 89.2 and 76.0 %, or 85.4 and 80.1 %, respectively. Of the three algorithms applied to cohort data and validated in the study, two indicated 100 % sensitivity in detecting cervical cancer and a low rate of referrals for colposcopy. CONCLUSIONS: These algorithms might contribute to precise and objective cervical cancer diagnostics in the outpatient departments of hospitals in countries with high mortality and low screening rates or areas with uneven resource distribution.