Diagnosis of malignant body fluids via cancer-universal methylation in cell-free DNA.

BACKGROUNDDifferentiating malignant from nonmalignant body fluids remains a clinical challenge because of the unsatisfying performance of conventional cytology. We aimed to improve the sensitivity and ubiquity of cancer cell detection by assaying universal cancer-only methylation (UCOM) markers in supernatant cell-free DNA (cfDNA).METHODSAn observational prospective cohort including 1,321 nonmalignant and malignant body fluids of multiple cancers was used to develop and validate a cfDNA UCOM methylation diagnostic assay. All samples were divided into 2 portions for cytology and supernatant cfDNA methylation analysis.RESULTSThe significant hypermethylation of a potentially novel UCOM marker, TAGMe, together with the formerly reported PCDHGB7, was identified in the cfDNA of malignant body fluid samples. The combined model, cell-free cancer-universal methylation (CUE), was developed and validated in a prospective multicancer cohort with markedly elevated sensitivity and specificity, and was further verified in a set containing additional types of malignant body fluids and metastases. In addition, it remained hypersensitive in detecting cancer cells in cytologically negative malignant samples.CONCLUSIONcfDNA methylation markers are robust in detecting tumor cells and are applicable to diverse body fluids and tumor types, providing a feasible complement to current cytology-based diagnostic analyses.TRIAL REGISTRATIONThis study was registered at Chictr.org.cn (ChiCTR2200060532).FUNDINGNational Natural Science Foundation of China (32270645, 31872814, 32000505, 82170088), the National Key R&D Program of Ningxia Hui Autonomous region (2022BEG01003), Shanghai Municipal Key Clinical Specialty (shslczdzk02201), Science and Technology Commission of Shanghai Municipality (20DZ2261200, 20DZ2254400), and Major Special Projects of Basic Research of Shanghai Science and Technology Commission (18JC1411101).

PCDHGB7 hypermethylation-based Cervical cancer Methylation (CerMe) detection for the triage of high-risk human papillomavirus-positive women: a prospective cohort study.

BACKGROUND: Implementation of high-risk human papillomavirus (hrHPV) screening has greatly reduced the incidence and mortality of cervical cancer. However, a triage strategy that is effective, noninvasive, and independent from the subjective interpretation of pathologists is urgently required to decrease unnecessary colposcopy referrals in hrHPV-positive women. METHODS: A total of 3251 hrHPV-positive women aged 30-82 years (median = 41 years) from International Peace Maternity and Child Health Hospital were included in the training set (n = 2116) and the validation set (n = 1135) to establish Cervical cancer Methylation (CerMe) detection. The performance of CerMe as a triage for hrHPV-positive women was evaluated. RESULTS: CerMe detection efficiently distinguished cervical intraepithelial neoplasia grade 2 or worse (CIN2 +) from cervical intraepithelial neoplasia grade 1 or normal (CIN1 -) women with excellent sensitivity of 82.4% (95% CI = 72.6 ~ 89.8%) and specificity of 91.1% (95% CI = 89.2 ~ 92.7%). Importantly, CerMe showed improved specificity (92.1% vs. 74.9%) in other 12 hrHPV type-positive women as well as superior sensitivity (80.8% vs. 61.5%) and specificity (88.9% vs. 75.3%) in HPV16/18 type-positive women compared with cytology testing. CerMe performed well in the triage of hrHPV-positive women with ASC-US (sensitivity = 74.4%, specificity = 87.5%) or LSIL cytology (sensitivity = 84.4%, specificity = 83.9%). CONCLUSIONS: PCDHGB7 hypermethylation-based CerMe detection can be used as a triage strategy for hrHPV-positive women to reduce unnecessary over-referrals. TRIAL REGISTRATION: ChiCTR2100048972. Registered on 19 July 2021.

Integrated Transcriptomic and Epigenetic Study of PCOS: Impact of Map3k1 and Map1lc3a Promoter Methylation on Autophagy.

Polycystic ovary syndrome (PCOS) is a prevalent heterogeneous endocrine and metabolic disorder in women of reproductive age. Epigenetic mechanisms contribute to the development of PCOS. Nevertheless, the role of DNA methylation in the development of PCOS remains unclear. To investigate the molecular mechanisms underlying the hyperandrogenic phenotype of PCOS, dihydrotestosterone (DHT)-induced prenatally androgenized (PNA) mice were used to mimic this phenotype. Ovarian samples from PNA and control mice were subjected to methyl-CpG-binding domain (MBD)-seq and RNA-seq, and validation was conducted using methylation-specific polymerase chain reaction (MSP) and quantitative real-time PCR (RT-qPCR). Immunohistochemical analysis (using anti-LC3II antibody) and transmission electron microscopy were conducted using ovarian tissue sections (which included granulosa cells) from PNA and control mice. There were 857 genes with differentially methylated promoter regions and 3,317 differentially expressed genes (DEGs) in the PNA mice compared to the control mice. Downregulation of Dnmt1 (which encodes DNA methyltransferase 1), accompanied by global hypomethylation, was observed in the PNA mice compared to the control mice. The promoter regions of Map3k1 (which encodes MEKK1) and Map1lc3a (which encodes LC3II) were hypomethylated, accompanied by upregulation of Map3k1 and Map1lc3a mRNA expression. The autophagy profiling results showed that LC3II protein expression and autophagosomes were significantly increased in the granulosa cells of PNA mice. Additionally, the mRNA expression of genes related to the mitogen-activated protein kinase (MAPK)/p53 pathway (Mapk14, Mapkapk3, and Trp53) and the autophagy-related gene Becn1 were significantly increased. DHT could change the DNA methylation and transcription level of Map3k1 and lead to an activation of autophagy in granulosa cells. These observations indicated that the change in autophagy may be driven by MAPK/p53 pathway activation, which may have been caused by DHT-induced transcriptional, and the methylation level changed of the key upstream gene Map3k1. Our study provides a novel genetic basis and new insights regarding the pathogenesis of PCOS.

Methylome and transcriptome profiling revealed epigenetic silencing of LPCAT1 and PCYT1A associated with lipidome alterations in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine diseases of fertile women and a major cause of infertility. The regulatory effects of DNA methylation on gene transcription and downstream lipid metabolism have not been explored in PCOS. In this study, MBD-seq and RNA-seq were performed on ovarian granulosa cells of PCOS patients and controls, and methylation specific PCR and quantitative polymerase chain reaction were used to validate the results. Then lipidomic profiling was conducted on serum of PCOS patients and controls using UPLC-MS. We identified 73 genes with differently methylated promoters and 830 differently expressed genes. The promoter regions of LPCAT1 and PCYT1A were hypermethylated, accompanied by downregulation of their messenger RNA expression, which may be involved in the regulation of PCOS through downstream glycerophospholipid metabolism and phosphatidylcholine synthesis. The lipid profiling results showed significant changes in 21 lipids, which demonstrated the disturbance in glycerophospholipid metabolism and glycerolipid metabolism pathways. Furthermore, the metabolites-genes interaction network was constructed to illustrate the association of aberrant methylome and transcriptome with lipidome alterations in glycerolipid and glycerophospholipid metabolism pathways. Our study suggested that the methylation silencing of LPCAT1 and PCYT1A may promote glycerophospholipids metabolism dysregulation, which provided a novel genetic and lipometabolic basis for the pathogenesis of PCOS.

Hypermethylated PCDHGB7 as a Biomarker for Early Detection of Endometrial Cancer in Endometrial Brush Samples and Cervical Scrapings.

Endometrial cancer (EC) is one of the most common gynecologic cancers in developed countries. Presently, it is imperative to develop a reliable, noninvasive, or minimally invasive detection method for EC. We explored the possibility of using DNA methylation marker from endometrial brush samples (with a "Tao brush") and cervical scrapes (with a "Pap brush") for early detection of EC. We analyzed the methylation data of EC and normal endometrial tissues from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data sets. An optimized methylation-sensitive restriction enzyme combined with real-time fluorescent quantitative PCR (MSRE-qPCR) was used for methylation detection. Included in the training set were 143 endometrial tissues, 103 Tao, and 109 Pap brush samples. The validation set included 110 Tao and 112 Pap brush samples. PCDHGB7 was significantly hypermethylated in EC compared with normal endometrial tissues in the TCGA and GEO data sets (AUC >0.95), which was verified in clinical samples. In the Pap brush samples, the AUC was 0.86 with 80.65% sensitivity and 82.81% specificity, whereas the Tao brush samples exhibited higher specificity (95.31%). The combination of Tao and Pap brush samples significantly increased the sensitivity to 90.32%. In the validation set, the final model yielded a sensitivity of 98.61%, specificity of 60.53%, positive predictive value of 82.56%, and negative predictive value of 95.83%. These results demonstrate the potential application of the novel methylation marker, hypermethylated PCDHGB7, in cervical scrapings and endometrial brush, which provides a viable, noninvasive, or minimally invasive method for early endometrial cancer detection across different clinical features and histologies to supplement current hysteroscopy diagnosis.

Identification of epigenetic interactions between microRNA and DNA methylation associated with polycystic ovarian syndrome.

Aberration in microRNA expression or DNA methylation is a causal factor for polycystic ovarian syndrome. However, the epigenetic interactions between miRNA and DNA methylation remain unexplored in PCOS. We conducted a novel integrated analysis of RNA-seq, miRNA-seq, and methylated DNA-binding domain sequencing on ovarian granulosa cells to reveal the epigenetic interactions involved in the pathogenesis of PCOS. We identified 830 genes and 30 miRNAs that were expressed differently in PCOS, and seven miRNAs negatively regulated target mRNA expression. 130 miRNAs' promoters were significantly differently methylated, while 13 were associated with miRNA expression. Furthermore, the hypermethylation of miR-429, miR-141-3p, and miR-126-3p' promoter was found related to miRNA expression suppression and therefore their corresponding genes upregulation, including XIAP, BRD3, MAPK14, and SLC7A5. Our findings provide a novel insight in PCOS. The consequential reversal of genes silencing may participate in PCOS pathogenesis and served as potential molecular targets for PCOS.

Post-Transcriptional Dysregulation of microRNA and Alternative Polyadenylation in Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide. microRNAs (miRNAs) repress gene expression by binding to complementary sequences in the 3' untranslated region (3'UTR) of target mRNAs. Alternative polyadenylation (APA) are relevant to the variability of the 3'UTR of mRNA. However, the posttranscriptional dysregulation of miRNAs and APA in CRC are poorly understood. METHOD: In this study, we conducted small RNA sequencing to identify differentially expressed miRNAs (DERs) and their target genes. Function analysis on DER-target genes can explain the regulation roles of miRNAs in CRC. The mutual regulation of miRNAs and APA was analyzed by combining miRNA data to 3'UTR alteration using 3' termini of polyadenylated RNAs sequencing (3T-seq) technique, and this was validated using TCGA gene expression data. RESULTS: Our results showed 64 significant differentially expressed miRNAs (DERs) in CRC patients. Their target genes were related to cell adhesion and transcription regulation and were prevailingly involved in the CRC-related pathway. Integrative analysis of the miRNA and APA profile revealed 16 DERs were correlated with 12 polyadenylation factors, and six of them were significantly differently expressed in CRC. We also found four DERs that lost binding sites due to APA and showed a positive correlation between the miRNA and gene expression. CONCLUSION: Our study found that miRNAs regulated APA by modulating key polyadenylation factors, and several miRNAs lost their suppression on mRNA due to APA. Associating this with gene expression may provide some important clues for a deeper study of posttranscriptional cellular regulation and biomarker research in CRC. Our data provided the first evidence that the interaction between miRNAs and APA associated with gene expression could serve as biomarkers for CRC, suggesting that hsa-miR-133a-3p and MLEC, hsa-miR-145-5p and SET, hsa-miR-1-3p and PPIA, and hsa-miR-378d and YY1 might be novel and potential biomarkers in improving the diagnosis of CRC.