Epigenetic upregulation of TET2 is an independent poor prognostic factor for intrahepatic cholangiocarcinoma

Mutations in IDH1/2 and the epigenetic silencing of TET2 occur in leukaemia or glioma in a mutually exclusive manner. Although intrahepatic cholangiocarcinoma (iCCA) may harbour IDH1/2 mutations, the contribution of TET2 to carcinogenesis remains unknown. In the present study, the expression and promoter methylation of TET2 were investigated in iCCA. The expression of TET2 was assessed in 52 cases of iCCA (small-duct type, n = 33; large-duct type, n = 19) by quantitative PCR, immunohistochemistry (IHC) and a sequencing-based methylation assay, and its relationships with clinicopathological features and alterations in cancer-related genes (e.g., KRAS and IDH1) were investigated. In contrast to non-neoplastic bile ducts, which were negative for TET2 on IHC, 42 cases (81%) of iCCA showed the nuclear overexpression of TET2. Based on IHC scores (area × intensity), these cases were classified as TET2-high (n = 25) and TET2-low (n = 27). The histological type, tumour size, lymph node metastasis and frequency of mutations in cancer-related genes did not significantly differ between the two groups. Overall and recurrence-free survival were significantly worse in patients with TET2-high iCCA than in those with TET2-low iCCA. A multivariate analysis identified the high expression of TET2 as an independent prognostic factor (HR = 2.94; p = 0.007). The degree of methylation at two promoter CpG sites was significantly less in TET2-high iCCA than in TET2-low iCCA or non-cancer tissue. In conclusion, in contrast to other IDH-related neoplasms, TET2 overexpression is common in iCCA of both subtypes, and its high expression, potentially induced by promoter hypomethylation, is an independent poor prognostic factor.

Comprehensive DNA Methylation Analysis of Human Neuroblastoma Cells Treated With Haloperidol and Risperidone

Accumulating evidence suggests that the epigenetic alterations induced by antipsychotics contribute to the therapeutic efficacy. However, global and site-specific epigenetic changes by antipsychotics and those shared by different classes of antipsychotics remain poorly understood. We conducted a comprehensive DNA methylation analysis of human neuroblastoma cells cultured with antipsychotics. The cells were cultured with low and high concentrations of haloperidol or risperidone for 8 days. DNA methylation assay was performed with the Illumina HumanMethylation450 BeadChip. We found that both haloperidol and risperidone tended to cause hypermethylation changes and showed similar DNA methylation changes closely related to neuronal functions. A total of 294 differentially methylated probes (DMPs), including 197 hypermethylated and 97 hypomethylated DMPs, were identified with both haloperidol and risperidone treatment. Gene ontology analysis of the hypermethylated probe-associated genes showed enrichment of genes related to the regulation of neurotransmitter receptor activity and lipoprotein lipase activity. Pathway analysis identified that among the DMP-associated genes, SHANK1 and SHANK2 were the major genes in the neuropsychiatric disorder-related pathways. Our data would be valuable for understanding the mechanisms of action of antipsychotics from an epigenetic viewpoint.

Multiple Biomarker-Combined Screening for Colorectal Cancer Based on Bisulfate Conversion-Free Detection of Fecal DNA Methylation

To evaluate the applicability of bisulfate conversion-free methylation assay based on enzyme digestion in fecal screening for colorectal cancer (CRC). Stool samples were collected from a total of 1142 participants with intestinal abnormalities, including 180 positive cases, 60 advanced adenomas, and 902 negative cases. DNA from reference cell lines and clinical samples was extracted and digested with an enzyme to detect the methylation of CRC markers SEPT9, SDC2, NDRG4, SFRP2, and BMP3 genes. Statistical analysis was then used to determine the ability of the markers, both individually and in combination, to detect CRC and adenoma. Our results showed that the enzyme digestion method could suitably detect DNA marker methylation in as low as 1% of the cell lines. BMP3 had a considerably low detection rate in all clinical samples, with only 6 positive cases detected out of 180 cancer samples. Our findings showed that the combination of SEPT9, SDC2, and SFRP2 had an area under the receiver operation curve of 0.937, sensitivity of 94.11%, and specificity of 89.21% for detecting CRC. Moreover, the detection sensitivity of adenoma can also reach 38.33%. After innovatively utilizing bisulfate conversion-free methylation assay for CRC screening, this study verified the potential clinical applicability of combining multiple biomarkers for CRC screening in a large number of samples.

METTL3 Intensifies the Progress of Oral Squamous Cell Carcinoma via Modulating the m6A Amount of PRMT5 and PD-L1

N6-Methyladenosine (m6A) modification is one of the commonest chemical modifications in eukaryotic mRNAs, which has essential effects on mRNA translation, splicing, and stability. Currently, there is a rising concern on the regulatory role of m6A in tumorigenesis. As a known component in the m6A methyltransferase complex, METTL3 (methyltransferase-like 3) plays an essential role in m6A methylation. Till now, the functions of METTL3 in oral squamous cell carcinoma (OSCC) and its relative mechanism remain to be explored. In this research, through the GEPIA database, we found that high METTL3 expression has a correlation with poor prognosis of squamous cell carcinoma of head and neck. qRT-PCR displayed that METTL3 was highly expressed in OSCC cells. Functionally, METTL3 knockdown reduced the invasion, migration, and proliferation competence of OSCC cells and attenuated the activation of CD8+ T cells. In contrast, METTL3 overexpression resulted in opposite results. GEPIA, UALCAN, and SRAMP databases, PCR, western blot, and m6A RNA methylation assay confirmed the m6A modification of PRMT5 and PD-L1 mediated by METTL3. In conclusion, our results displayed that METTL3 intensified the metastasis and proliferation of OSCC by modulating the m6A amounts of PRMT5 and PD-L1, suggesting that METTL3 may be a therapeutic target for OSCC patients.

A urine-based DNA methylation assay to facilitate early detection and risk stratification of bladder cancer

Background Current non-invasive tests have limited sensitivities and lack capabilities of pre-operative risk stratification for bladder cancer (BC) diagnosis. We aimed to develop and validate a urine-based DNA methylation assay as a clinically feasible test for improving BC detection and enabling pre-operative risk stratifications. Methods A urine-based DNA methylation assay was developed and validated by retrospective single-center studies in patients of suspected BC in Cohort 1 (n = 192) and Cohort 2 (n = 98), respectively. In addition, a prospective single-center study in hematuria patient group (Cohort 3, n = 174) was used as a second validation of the model. Results The assay with a dual-marker detection model showed 88.1% and 91.2% sensitivities, 89.7% and 85.7% specificities in validation Cohort 2 (patients of suspected BC) and Cohort 3 (patients of hematuria), respectively. Furthermore, this assay showed improved sensitivities over cytology and FISH on detecting low-grade tumor (66.7–77.8% vs. 0.0–22.2%, 0.0–22.2%), Ta tumor (83.3% vs. 22.2–41.2%, 44.4–52.9%) and non-muscle invasive BC (NMIBC) (80.0–89.7% vs. 51.5–52.0%, 59.4–72.0%) in both cohorts. The assay also had higher accuracies (88.9–95.8%) in diagnosing cases with concurrent genitourinary disorders as compared to cytology (55.6–70.8%) and FISH (72.2–77.8%). Meanwhile, the assay with a five-marker stratification model identified high-risk NMIBC and muscle invasive BC with 90.5% sensitivity and 86.8% specificity in Cohort 2. Conclusions The urine-based DNA methylation assay represents a highly sensitive and specific approach for BC early-stage detection and risk stratification. It has a potential to be used as a routine test to improve diagnosis and prognosis of BC in clinic.

Promoter Methylation and Gene Expression of Pin1 Associated with the Risk of Alzheimer’s Disease in Southern Chinese

Background: Pin1 is a propyl cis-trans isomerase and it has been associated with age-atonset of Alzheimer’s disease (AD) and other pathological characteristics of AD. DNA methylation is one of the gene regulation mechanisms and it might affect the gene expression. Objectives: This study was aimed to examine the correlation between DNA methylation and gene expression of Pin1 and its effect on the risk of AD in a Chinese population. Methods: 80 AD patients and 180 normal controls were recruited in this study and their cognitive functions were assessed. Pin1 gene expression and methylation were quantified by real-time RT-PCR and Melting Curve Analysis-Methylation assay (MCA-Meth), respectively. Results: Our finding revealed a positive correlation between methylation and gene expression of Pin1 (p=0.001) and increased Pin1 methylation was predisposed to the risk of AD (p<0.001). CG genotype of Pin1 SNP rs2287839 was associated with higher gene expression of Pin1 (p=0.036) and the effect was only prominent in normal controls as AD patients were already methylated at Pin1 promoter. Furthermore, methylation of Pin1 was associated with better performance in cognition (p=0.018). Conclusion: Our result further supported the involvement of Pin1 in AD and the increased level of Pin1 might be a protective factor for AD.