A Genome-Wide Investigation of Effects of Aberrant DNA Methylation on the Usage of Alternative Promoters in Hepatocellular Carcinoma

BackgroundThe alternative usage of promoters provides a way to regulate gene expression, has a significant influence on the transcriptome, and contributes to the cellular transformation of cancer. However, the function of alternative promoters (APs) in hepatocellular carcinoma (HCC) has not been systematically studied yet. In addition, the potential mechanism of regulation to the usage of APs remains unclear. DNA methylation, one of the most aberrant epigenetic modifications in cancers, is known to regulate transcriptional activity. Whether DNA methylation regulates the usage of APs needs to be explored. Here, we aim to investigate the effects of DNA methylation on usage of APs in HCC.MethodsPromoter activities were calculated based on RNA-seq data. Functional enrichment analysis was implemented to conduct GO terms. Correlation tests were used to detect the correlation between promoter activity and methylation status. The LASSO regression model was used to generate a diagnostic model. Kaplan–Meier analysis was used to compare the overall survival between high and low methylation groups. RNA-seq and whole-genome bisulfite sequencing (WGBS) in HCC samples were performed to validate the correlation of promoter activity and methylation.ResultsWe identified 855 APs in total, which could be well used to distinguish cancer from normal samples. The correlation of promoter activity and DNA methylation in APs was observed, and the APs with negative correlation were defined as methylation-regulated APs (mrAPs). Six mrAPs were identified to generate a diagnostic model with good performance (AUC = 0.97). Notably, the majority of mrAPs had CpG sites that could be used to predict clinical outcomes by methylation status. Finally, we verified 85.6% of promoter activity variation and 92.3% of methylation changes in our paired RNA-seq and WGBS samples, respectively. The negative correlation between promoter activity and methylation status was further confirmed in our HCC samples.ConclusionThe aberrant methylation status plays a critical role in the precision usage of APs in HCC, which sheds light on the mechanism of cancer development and provides a new insight into cancer screening and treatment.

GLAD-PCR assay of DNA methylation sites in regulatory regions of some tumor-suppressor genes in breast cancer

Hypermethylation of the RcgY sites is shown for many cancer diseases. such aberrant methylation, suppressing the gene activity, occurs at early stages of carcinogenesis. Recently, using glad-pcR assay, we have detected aberrantly methylated RcgY sites, which can be considered to be epigenetic markers of colorectal, lung, and gastric cancers. in breast cancer, methylation of the regulatory regions of ALX4, BMP2, CCND2, CDH13, CDX1, FOXA1, GALR1, GATA5, GREM1, HIC1, HMX2, HS3ST2, HOXC10, ICAM5, LAMA1, RARB, RASSF1A, RUNX3, RXRG, RYR2, SFRP2, SOX17, TERT, and ZNF613 tumor-suppressor genes is reported. in the present work, we determined aberrantly methylated RcgY sites in the regulatory regions of these genes in dNa preparations from breast cancer tissues. the study of dNa samples from 30 tumor and 22 normal mammary tissue samples demonstrates a high diagnostic potential of selected R(5mc)gY sites in regulatory regions of CCND2, BMP2, GALR1, SOX17, HMX2, and HS3ST2 genes with total index of sensitivity and specificity for R(5mc)gY detection in tumor dNa 90.0 % and 100.0 %, respectively.

Genome-wide aberrant methylation in primary metastatic UM and their matched metastases

Uveal melanoma (UM) is an aggressive intra-ocular cancer with a strong tendency to metastasize. Metastatic UM is associated with mutations in BAP1 and SF3B1, however only little is known about the epigenetic modifications that arise in metastatic UM. In this study we aim to unravel epigenetic changes contributing to UM metastasis using a new genome-wide methylation analysis technique that covers over 50% of all CpG’s. We identified aberrant methylation contributing to BAP1 and SF3B1-mediated UM metastasis. The methylation data was integrated with expression data and surveyed in matched UM metastases from the liver, skin and bone. UM metastases showed no commonly shared novel epigenetic modifications, implying that epigenetic changes contributing to metastatic spreading and colonization in distant tissues occur early in the development of UM and epigenetic changes that occur after metastasis are mainly patient-specific. Our findings reveal a plethora of epigenetic modifications in metastatic UM and its metastases, which could subsequently result in aberrant repression or activation of many tumor-related genes. This observation points towards additional layers of complexity at the level of gene expression regulation, which may explain the low mutational burden of UM.

Aberrant Methylation of SLIT2 Gene in Plasma Cell-Free DNA of Non-Small Cell Lung Cancer Patients

This study aimed to understand aberrant methylation of SLITs genes as a biomarker for the early detection and prognosis prediction of non-small cell lung cancer (NSCLC). Methylation levels of SLITs were determined using the Infinium HumanMethylation450 BeadChip or pyrosequencing. Five CpGs at the CpG island of SLIT1, SLIT2 or SLIT3 genes were significantly (Bonferroni corrected p < 0.05) hypermethylated in tumor tissues obtained from 42 NSCLC patients than in matched normal tissues. Methylation levels of these CpGs did not differ significantly between bronchial washings obtained from 76 NSCLC patients and 60 cancer-free patients. However, methylation levels of SLIT2 gene were significantly higher in plasma cell-free DNA of 72 NSCLC patients than in that of 61 cancer-free patients (p = 0.001, Wilcoxon rank sum test). Prediction of NSCLC using SLIT2 methylation was achieved with a sensitivity of 73.7% and a specificity of 61.9% in a plasma test dataset (N = 40). A Cox proportional hazards model showed that SLIT2 hypermethylation in plasma cell-free DNA was significantly associated with poor recurrence-free survival (hazards ratio = 2.19, 95% confidence interval = 1.21–4.36, p = 0.01). The present study suggests that aberrant methylation of SLIT2 in plasma cell-free DNA is a valuable biomarker for the early detection of NSCLC and prediction of recurrence-free survival. However, further research is needed with larger sample size to confirm results.

Pan-Cancer DNA Methylation Analysis and Tumor Origin Identification of Carcinoma of Unknown Primary Site Based on Multi-Omics

The metastatic cancer of unknown primary (CUP) sites remains a leading cause of cancer death with few therapeutic options. The aberrant DNA methylation (DNAm) is the most important risk factor for cancer, which has certain tissue specificity. However, how DNAm alterations in tumors differ among the regulatory network of multi-omics remains largely unexplored. Therefore, there is room for improvement in our accuracy in the prediction of tumor origin sites and a need for better understanding of the underlying mechanisms. In our study, an integrative analysis based on multi-omics data and molecular regulatory network uncovered genome-wide methylation mechanism and identified 23 epi-driver genes. Apart from the promoter region, we also found that the aberrant methylation within the gene body or intergenic region was significantly associated with gene expression. Significant enrichment analysis of the epi-driver genes indicated that these genes were highly related to cellular mechanisms of tumorigenesis, including T-cell differentiation, cell proliferation, and signal transduction. Based on the ensemble algorithm, six CpG sites located in five epi-driver genes were selected to construct a tissue-specific classifier with a better accuracy (&gt;95%) using TCGA datasets. In the independent datasets and the metastatic cancer datasets from GEO, the accuracy of distinguishing tumor subtypes or original sites was more than 90%, showing better robustness and stability. In summary, the integration analysis of large-scale omics data revealed complex regulation of DNAm across various cancer types and identified the epi-driver genes participating in tumorigenesis. Based on the aberrant methylation status located in epi-driver genes, a classifier that provided the highest accuracy in tracing back to the primary sites of metastatic cancer was established. Our study provides a comprehensive and multi-omics view of DNAm-associated changes across cancer types and has potential for clinical application.

Hypermethylation-mediated Transcriptional Silencing of lncRNA-SCARF1 Promotes Progression and Metastasis of Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related deaths. Recent studies have demonstrated that deregulation of long noncoding RNAs (lncRNAs), such as abnormal DNA methylation of promoter, is strongly associated with development and progression of diverse malignant tumors. This study investigated the mechanisms and changes in DNA methylation levels of promoter regions of HCC-specific lncRNAs, and alterations of downstream target genes.Methods: LncRNA expression profile data of 8 human HCC tissues and matched normal tissues were obtained. LncRNAs with aberrant methylation were identified through DNA methylation microarray. The biological functions of the lncRNAs were investigated through targeted knockdown of lncRNA-SCARF1 in vitro and in vivo. Furthermore, the downstream targets of lncRNA-SCARF1 were identified through ChIRP-MS.Results: LncRNA-SCARF1 was significantly down-regulated in HCC samples. Hypermethylation in the promoter of lnc-SCARF1 induced its down-regulation in HCC. Over-expression of lnc-SCARF1 inhibited the tumor proliferation and migration ability of HCC cells in vitro and in vivo. Furthermore, CUL9 was found to be a potential downstream target of lncRNA-SCARF1.Conclusion: LncRNA-SCARF1 regulates HCC progression by interacting with CUL9 and may serve as a prognostic biomarker or an effective therapeutic target in HCC.

Long-term Changes In Aberrant DNA Methylation And Gastritis After Helicobacter Pylori Eradication Focused On Metachronous Gastric Cancer

BackgroundA persistently high methylation level in gastric mucosa after Helicobacter pylori (H. pylori) eradication is presumed to be a risk for metachronous gastric cancer (MGC); however, long-term changes in aberrant DNA methylation and histological gastritis have been unclear. Our aim was to examine changes in DNA methylation and histological gastritis according to the occurrence of MGC.MethodsSubjects were classified into 3 groups: 25 patients in whom metachronous gastric cancers occurred after the initial endoscopic resection (ER) for early gastric cancer and H. pylori eradication (MGC group), 17 patients in whom MGC did not occur for more than 5 years after initial ER and H. pylori eradication (non-MGC group) and 29 patients without a history of gastric cancer who succeeded in eradication more than 5 years ago (HP group). Aberrance of DNA methylation in 3 genes (miR-124a-3, EMX1, NKX6-1) and histological score of atrophy and intestinal metaplasia (IM) were evaluated using biopsy samples before and more than a mean of 5 years after H. pylori eradication.ResultsThe methylation level of miR-124a-3 in the HP group and non-MGC group and that of EMX1 in the HP group significantly decreased in the long term after eradication. In the MGC group, H. pylori eradication did not improve aberrant methylation, and the Z-score significantly increased. There were significant positive correlations between methylation levels in miR-124a-3 and EMX1 and histological findings after eradication.ConclusionsA persistently high methylation level after H. pylori eradication reflected precancerous mucosal conditions and led to long-term MGC.

Deoxyribonucleic Acid 5-Hydroxymethylation in Cell-Free Deoxyribonucleic Acid, a Novel Cancer Biomarker in the Era of Precision Medicine

Aberrant methylation has been regarded as a hallmark of cancer. 5-hydroxymethylcytosine (5hmC) is recently identified as the ten-eleven translocase (ten-eleven translocase)-mediated oxidized form of 5-methylcytosine, which plays a substantial role in DNA demethylation. Cell-free DNA has been introduced as a promising tool in the liquid biopsy of cancer. There are increasing evidence indicating that 5hmC in cell-free DNA play an active role during carcinogenesis. However, it remains unclear whether 5hmC could surpass classical markers in cancer detection, treatment, and prognosis. Here, we systematically reviewed the recent advances in the clinic and basic research of DNA 5-hydroxymethylation in cancer, especially in cell-free DNA. We further discuss the mechanisms underlying aberrant 5hmC patterns and carcinogenesis. Synergistically, 5-hydroxymethylation may act as a promising biomarker, unleashing great potential in early cancer detection, prognosis, and therapeutic strategies in precision oncology.

Investigation of Isocitrate Dehydrogenase 1 and 2 Mutations in Acute Leukemia Patients in Saudi Arabia

Different forms of human cancer show mutations for isocitrate dehydrogenases 1 and 2 (IDH1/2). Mutation of these genes can cause aberrant methylation of the genome CpG islands (CGIs), which leads to an increase of suppressed oncogenes transcription or repression of active tumor suppressor gene transcription. This study aimed to identify the prevalence of IDH1/2 mutations in acute leukemia patients. The study cohort included 43 AML patients and 30 childhood ALL patients, from whom DNA bone marrow samples were taken. The alteration hotspots in codons IDH1 (R132) and IDH2 (R172 and R140) were examined via direct sequencing. Mutations in IDH1 were detected in 7 out of 43 (16.2%) AML patients; 5 of them occurred at codon R132. The other two mutations included a single-nucleotide polymorphism, which affected codon G105 in one patient. However, no mutation was detected in the IDH2 in any of the patients. Moreover, no mutations were detected in either IDH1 or IDH2 in ALL patients. The dominance of IDH1 mutations in AML, which was 16%, emphasizes the existence of the mutation in our population. On the other hand, IDH2 mutation was observed to be less frequent in both illnesses. Due to the limitation of using a small sample size, larger cohort screening is recommended to determine their usefulness as prognostic indicators.