Epigenetic Silencing of Tumor Suppressor lncRNA NKILA: Implication on NF-κB Signaling in Non-Hodgkin’s Lymphoma

The long non-coding RNA (lncRNA) NKILA, localized to 20q13.31, is a negative regulator of NF-κB signaling implicated in carcinogenesis. As a CpG island is embedded in the promoter region of NKILA, it is hypothesized as a tumor suppressor lncRNA silenced by promoter DNA methylation in non-Hodgkin’s lymphoma (NHL). By pyrosequencing-verified methylation-specific PCR, NKILA methylation was detected in 1/10 (10%) NHL cell lines, but not in normal peripheral blood buffy coats or tonsils. NKILA methylation correlated with the repression of NKILA in cell lines. Hypomethylation treatment with 5-Aza-2′-deoxycytidine resulted in promoter demethylation and the re-expression of NKILA. In 102 NHL primary samples, NKILA was methylated in 29 (51.79%) diffuse large B-cell lymphoma (DLBCL) and 4 (20%) peripheral T-cell lymphoma cases, but unmethylated in all 26 mantle cell lymphoma cases. Mechanistically, the knockdown of NKILA resulted in promoting IkBα phosphorylation, associated with nucleus translocation of total p65 and phosphorylated p65 in SU-DHL-1 cells, hence constitutive NF-κB activation. Functionally, the knockdown of NKILA in SU-DHL-1 cells led to decreased cell death and increased cellular proliferation. Collectively, NKILA was a tumor suppressor lncRNA frequently hypermethylated in DLBCL. Promoter DNA methylation-mediated NKILA silencing resulted in increased cellular proliferation and decreased cell death via the repression of NF-κB signaling in NHL.

Haploinsufficiency by minute MutL homolog 1 promoter DNA methylation may represent unique phenotypes of microsatellite instability-gastric carcinogenesis

Promoter DNA methylation of MutL homolog 1 (MLH1) is considered to play a causative role in microsatellite instability (MSI) carcinogenesis in primary gastric cancer, and a high MSI status is associated with treatment sensitivity to human cancers. Nevertheless, clinicopathological analysis is defective for MLH1 methylation status in a quantitative manner. We newly developed quantitative methylation specific PCR using a TaqMan probe and applied it to 138 patients with primary gastric cancer who underwent gastrectomy in addition to basic molecular features such as MSI, Epstein Barr virus, and other DNA methylation status. (1) In primary gastric cancer, median methylation value was 0.055, ranging from 0 to 124.3. First, MLH1 hypermethylation was strongly correlated with MSI-High/MSI-Low status and suppressed immunostaining (P < 0.0001). (2) The MLH1 hypermethylation was associated with advanced age (P = 0.0048), antral location (P = 0.0486), synchronous multiple gastric cancer (P = 0.0001), and differentiated histology (P = 0.028). (3) Log-rank plot analysis identified the most relevant cut-off value (0.23) to reflect gentle phenotypes in MLH1 hypermethylation cases (P = 0.0019), especially in advanced gastric cancer (P = 0.0132), which are designated as haploinsufficiency of MSI (MSI-haplo) phenotype in this study. (4) In synchronous multiple gastric cancer, MLH1 hypermethylation was not necessarily confirmed as field cancerization. (5) MSI-haplo defined by MLH1 methylation status represented distinct prognostic phenotype even after molecular classifications. MLH1 hypermethylation designated as MSI-haplo may represent unique prognostic phenotype during gastric carcinogenesis.

Sex-specific maternal programming of corticosteroid-binding globulin by predator odour

Predation is a key organizing force in ecosystems. The threat of predation may act to programme the endocrine hypothalamic–pituitary–adrenal axis during development to prepare offspring for the environment they are likely to encounter. Such effects are typically investigated through the measurement of corticosteroids (Cort). Corticosteroid-binding globulin (CBG) plays a key role in regulating the bioavailability of Cort, with only free unbound Cort being biologically active. We investigated the effects of prenatal predator odour exposure (POE) in mice on offspring CBG and its impact on Cort dynamics before, during and after restraint stress in adulthood. POE males, but not females, had significantly higher serum CBG at baseline and during restraint and lower circulating levels of Free Cort. Restraint stress was associated with reduced liver transcript abundance of SerpinA6 (CBG-encoding gene) only in control males. POE did not affect SerpinA6 promoter DNA methylation. Our results indicate that prenatal exposure to a natural stressor led to increased CBG levels, decreased per cent of Free Cort relative to total and inhibited restraint stress-induced downregulation of CBG transcription. These changes suggest an adaptive response to a high predator risk environment in males but not females that could buffer male offspring from chronic Cort exposure.

A panel of epigenetically dysregulated Wnt signaling pathway genes for non-invasive diagnosis of pediatric acute lymphoblastic leukemia

BACKGROUND: Genetic and epigenetic dysregulation of Wnt signaling pathway is widely linked up with abnormal proliferation and/or epithelial-to-mesenchymal transition, in different cancer cell types. OBJECTIVE: In the present research, we have tested whether promoter DNA methylation of a set of Wnt/non-Wnt genes such as [cadherin-2 (CDH2)], “present in circulation”, could serve as “bone-marrow biopsy surrogate” and help in diagnosing the status, sub-type or treatment outcome in pediatric acute lymphoblastic leukemia (ALL) patients. METHODS: Promoter DNA methylation was quantified in the bisulfite modified blood from the pediatric ALL patients (n= 86) in comparison with age-matched cancer-free subjects (n= 28), using real-time methylation specific PCR followed by rigorous statistical validations. RESULTS: The observed methylation index, sensitivity and specificity of selected molecular markers (viz., SALL1, WNT5α, LRP1b, CDH2) in patients’ liquid-biopsies was clinically significant showing high positive correlation in the pre-B ALL cases (p-value < 0.001). A substantial drop in promoter methylation signal of the follow-up/post-treatment patients was also noted (p-value < 0.001), which suggested an impending role of minimally invasive liquid-biopsy approach in the diagnosis and/or therapeutic monitoring of pediatric leukemia. CONCLUSIONS: Whilst the reported metadata provides useful insight into the plausible involvement of epigenetic glitches in leukemogensis, our findings strengthen the remarkable functional consequences of dysregulated Wnt signaling genes in the hematological malignancies besides offering a novel panel of epigenetic marks.

The Expression of Interleukin-1β Is Regulated by DNA Methylation in Microglia of Hippocampus to Participate Postoperative Cognitive Dysfunction in Aged Mice

BackgroundPostoperative cognitive dysfunction (POCD) is one of the common postoperative complications in the elderly. The main clinical manifestation is memory impairment, which can cause permanent damage and even dementia in severe cases. However, the pathogenesis of POCD is still unknown. Age and neuroinflammation are known to be closely related to its occurrence, while DNA methylation is very important for transcriptional silencing and neuroinflammation. Consequently, this study intended to establish a mouse model of POCD to explore the role of DNA methylation in regulating the expression of interleukin-1β which participated POCD in aged mice.MethodsPOCD model was established by exploratory laparotomy and evaluated by new object experiment and Y maze test. In addition, ELISA, RT-PCR, Western blotting, immunofluorescence, microglia isolation and flow cytometry methods were used to detect the inflammatory state of dorsal hippocampal after surgery. Moreover, MSP, MeDIP and IL-1β promoter DNA methylation sequencing were used to explore the regulation of DNA methylation on IL-1β in this model. Finally, Golgi staining and Western blotting were used to further explore the role of IL-1β in POCD and its possible mechanisms. ResultsCognitive impairment was observed in aged but not adult mice at 1 day after surgery. There was a significant correlation between the level of IL-1β in dorsal hippocampus and the performance of cognitive function. The microglia in the dorsal hippocampus was activated and the IL-1β promoter DNA methylation was decreased in the aged mice. The increased expression of IL-1β impaired synaptic plasticity and hippocampus-dependent memory formation. Intracerebroventricular administration of IL-1β receptor antagonist could prevent the cognitive impairment of aged mice after surgery, reverse the decrease of dendritic spine density and synapse-associated protein expression induced by surgery.ConclusionDNA methylation regulation may be an important mechanism for greater susceptibility to POCD in aged mice by regulating the expression of IL-1β. IL-1β inhibiting prevented surgery-induced cognitive decline and synaptic plasticity dysfunction. The research also provided a new target for the clinical prevention of the occurrence of POCD in the elderly.