Methylation Regulation of TLR3 on Immune Parameters in Lung Adenocarcinoma

This study aims to analyze the methylation regulation of TLR3 in lung adenocarcinoma (LUAD) and to explore the association of TLR3 expression with immune microenvironment. TLR3 has a decreased expression in LUAD tissues and low expression of TLR3 is not only associated with poor prognosis in patients with LUAD, but also can be used as a diagnostic marker. Bisulfite sequencing PCR (BSP) results showed that the methylation level in the promoter of TLR3 was negatively correlated with the level of TLR3 mRNA in LUAD tissues. TIMER analysis showed that TLR3 was negatively correlated with the tumor purity of LUAD and positively with immune cell infiltration to some extent. ESTIMATE analysis also suggested that TLR3 expression and its methylation had significant correlation with immune score. The lower immune scores were associated with the late stage of LUAD and poor prognosis. The high expression of TLR3 might inhibit the development of LUAD by activating apoptosis pathway. The proteins interacted with TLR3 were mainly involved in the apoptosis pathway and positively correlated with the key genes (MYD88, Caspase 8, BIRC3, PIK3R1) in this pathway. Therefore, TLR3 as a key biomarker for prognosis and diagnosis in LUAD, might be considered as a potential epigenetic and immunotherapeutic target.

Profiles of mitochondrial DNA methylation in the ridgetail white prawn, Exopalaemon carinicauda (Holthuis, 1950) (Decapoda, Palaemonidae) under heavy metal stress from chromium and cadmium

For the purpose of studying the epigenetic characteristics of the mitochondrial genome of the ridgetail white prawn, Exopalaemon carinicauda (Holthuis, 1950) under Cd2+ and Cr6+ heavy metal stress, the mitochondrial DNA methylation of E. carinicauda was analysed by bisulfite sequencing PCR (BSP). Many methylation sites were found at the 3′ end sequence of COX3 and at the starting region sequence of ND3, while only a few methylation sites were found at the 3′ end sequence of ND5. The mitochondrial genome was inferred to regulate the energy metabolism through the methylation process. In addition, under Cd2+ stress, mitochondrial DNA methylation was more common, and found during all stress periods (3, 6, 12, 24, 48, 72 and 96 h), while under Cr6+ stress, mitochondrial DNA methylation was less common, mainly occurring after 48 hours of stress. The sensitivity of the mitochondrial genome response to Cd2+ stress was inferred to be greater than that to Cr6+. This study revealed for the first time that methylation occurs in the mitochondrial genome of E. carinicauda in response to heavy metal stress.

DNA Methylation Profiles and Their Diagnostic Utility in BC

Biomarkers, including DNA methylation, have shown a great potential for use in personalized medicine for BC and especially for the diagnosis of BC in developing countries. According to the bisulfite sequencing PCR in twelve specimens (BC and matched normal tissues), nine genetic probes were designed to detect the frequency of methylation of the promoters in a total of 302 paired cases of BC and matched normal breast tissues. Finally, a total of 900 serum samples were used to validate the use of these methylation biomarkers for clinical diagnosis of BC. A high frequency of promoter methylation of SFN, HOXA11, P16, RARβ, PCDHGB7, hMLH1, WNT5a, HOXD13, and RASSF1a was observed in BC tissues. The methylation frequencies of HOXD13 and hMLH1 increased with the progression of BC. The methylation frequencies of HOXD13 and WNT5a were significantly higher in BC. We found that methylation modification-positive samples were most consistently associated with luminal BC. Finally, we confirmed that RASSF1a, P16, and PCDHGB7 displayed a significant sensitivity and specificity as diagnostic biomarkers for BC (P<0.001), and a panel that combined these three genes displayed increased significance (AUC, 0.781; P<0.001). These data suggest that epigenetic markers in serum can potentially be used to diagnose BC. The identification of additional BC-specific methylated genes would improve the sensitivity and specificity of this approach. This study could also indicate that different molecular subtypes of BC are caused by distinct genetic and epigenetic mechanisms.

Different EPHX1 methylation levels in promoter area between carbamazepine-resistant epilepsy group and carbamazepine-sensitive epilepsy group in Chinese Population

Background: Epigenetics underlying refractory epilepsy is poorly understood. DNA methylation may affect gene expression in epilepsy patients without affecting DNA sequences. Herein, we investigated the association between Carbamazepine-resistant (CBZ-resistant) epilepsy and EPHX1 methylation in a northern Han Chinese population, and conducted an analysis of clinical risk factors for CBZ-resistant epilepsy. Methods: 75 northern Han Chinese patients participated in this research. 25 cases were CBZ-resistant epilepsy, 25 cases were CBZ-sensitive epilepsy and the remaining 25 cases were controls. Using a CpG searcher was to make a prediction of CpG islands; bisulfite sequencing PCR (BSP) was applied to test the methylation of EPHX1. We then did statistical analysis between clinical parameters and EPHX1 methylation. Results: There was no difference between CBZ-resistant patients,CBZ-sensitive patients and healthy controls in matched age and gender. However, a significant difference of methylation levels located in NC_000001.11 (225806929.....225807108) of the EPHX1 promoter was found in CBZ-resistant patients, which was much higher than CBZ-sensitive and controls. Additionally, there was a significant positive correlation between seizure frequency, disease course and EPHX1 methylation in CBZ-resistant group. Conclusion: Methylation levels in EPHX1 promoter associated with CBZ-resistant epilepsy significantly. EPHX1 methylation may be the potential marker for CBZ resistance prior to the CBZ therapy and potential target for treatments.

Different EPHX1 methylation levels in promoter area between carbamazepine-resistant epilepsy group and carbamazepine-sensitive epilepsy group in Chinese Population

Background: Epigenetics underlying refractory epilepsy is poorly understood. DNA methylation may affect gene expression in epilepsy patients without affecting DNA sequences. Herein, we investigated the association between Carbamazepine-resistant (CBZ-resistant) epilepsy and EPHX1 methylation in a northern Han Chinese population, and conducted an analysis of clinical risk factors for CBZ-resistant epilepsy. Methods: 75 northern Han Chinese patients participated in this research. 25 cases were CBZ-resistant epilepsy, 25 cases were CBZ-sensitive epilepsy and the remaining 25 cases were controls. Using a CpG searcher was to make a prediction of CpG islands; bisulfite sequencing PCR (BSP) was applied to test the methylation of EPHX1. We then did statistical analysis between clinical parameters and EPHX1 methylation. Results: There was no difference between CBZ-resistant patients,CBZ-sensitive patients and healthy controls in matched age and gender. However, a significant difference of methylation levels located in NC_000001.11 (225806929.....225807108) of the EPHX1 promoter was found in CBZ-resistant patients, which was much higher than CBZ-sensitive and controls. Additionally, there was a significant positive correlation between seizure frequency, disease course and EPHX1 methylation in CBZ-resistant group. Conclusion: Methylation levels in EPHX1 promoter associated with CBZ-resistant epilepsy significantly. EPHX1 methylation may be the potential marker for CBZ resistance prior to the CBZ therapy and potential target for treatments.

Different EPHX1 methylation levels in promoter area between carbamazepine-resistant epilepsy group and carbamazepine-sensitive epilepsy group in Chinese Population

Background: Epigenetics underlying refractory epilepsy is poorly understood. DNA methylation may affect gene expression in epilepsy patients without affecting DNA sequences. Herein, we investigated the association between Carbamazepine-resistant (CBZ-resistant) epilepsy and EPHX1 methylation in a northern Han Chinese population, and conducted an analysis of clinical risk factors for CBZ-resistant epilepsy. Methods: 75 northern Han Chinese patients participated in this research. 25 cases were CBZ-resistant epilepsy, 25 cases were CBZ-sensitive epilepsy and the remaining 25 cases were controls. Using a CpG searcher was to make a prediction of CpG islands; bisulfite sequencing PCR (BSP) was applied to test the methylation of EPHX1. We then did statistical analysis between clinical parameters and EPHX1 methylation. Results: There was no difference between CBZ-resistant patients,CBZ-sensitive patients and healthy controls in matched age and gender. However, a significant difference of methylation levels located in NC_000001.11 (225806929.....225807108) of the EPHX1 promoter was found in CBZ-resistant patients, which was much higher than CBZ-sensitive and controls. Additionally, there was a significant positive correlation between seizure frequency, disease course and EPHX1 methylation in CBZ-resistant group. Conclusion: Methylation levels in EPHX1 promoter associated with CBZ-resistant epilepsy significantly. EPHX1 methylation may be the potential marker for CBZ resistance prior to the CBZ therapy and potential target for treatments.

Different EPHX1 methylation levels in promoter area between carbamazepine-resistant epilepsy group and carbamazepine-sensitive epilepsy group in Chinese Population

Background: Epigenetics underlying refractory epilepsy is poorly understood. DNA methylation may affect gene expression in epilepsy patients without affecting DNA sequences. Herein, we investigated the association between Carbamazepine-resistant (CBZ-resistant) epilepsy and EPHX1 methylation in a northern Han Chinese population, and conducted an analysis of clinical risk factors for CBZ-resistant epilepsy. Methods: 75 northern Han Chinese patients participated in this research. 25 cases were CBZ-resistant epilepsy, 25 cases were CBZ-sensitive epilepsy and the remaining 25 cases were controls. Using a CpG searcher was to make a prediction of CpG islands; bisulfite sequencing PCR (BSP) was applied to test the methylation of EPHX1. We then did statistical analysis between clinical parameters and EPHX1 methylation. Results: There was no difference between CBZ-resistant patients,CBZ-sensitive patients and healthy controls in matched age and gender. However, a significant difference of methylation levels located in NC_000001.11 (225806929.....225807108) of the EPHX1 promoter was found in CBZ-resistant patients, which was much higher than CBZ-sensitive and controls. Additionally, there was a significant positive correlation between seizure frequency, disease course and EPHX1 methylation in CBZ-resistant group. Conclusion: Methylation levels in EPHX1 promoter associated with CBZ-resistant epilepsy significantly. EPHX1 methylation may be the potential marker for CBZ resistance prior to the CBZ therapy and potential target for treatments.

Down-Regulation of Laminin (LN)- α5 is Associated with Preeclampsia and Impairs Trophoblast Cell Viability and Invasiveness Through PI3K Signaling Pathway

Background/Aims: Preeclampsia (PE) is a gestational disorder defined as hypertension and proteinuria, which is deemed a major cause of maternal and neonatal mortality and morbidity worldwide. The aim of this study was to investigate the expression patterns of placental laminin (LN)-α5 expression in normal and PE pregnancies, as well as evaluating the effects of LN-α5 on trophoblast proliferation, apoptosis, and invasion. Methods: LN-α5 expression levels were examined by reverse-transcriptase polymerase chain reaction (RT-PCR), and further confirmed by western blotting and immunofluorescence staining. Cell proliferation and apoptosis were measured by CCK-8 assay and flow cytometry. Cell invasion was assessed by matrigel-based transwell assay. LN-α5 DNA methylation in placentas was determined by bisulfite sequencing PCR (BSP). Results: LN-α5 expression levels in PE placentas were significantly lower than that of normal pregnancies. Deficiency in LN-α5 expression resulted in decreased trophoblast proliferation and invasion but increased cell apoptosis, meanwhile, PI3K/AKT/mTOR signaling pathway was impaired by LN-α5 silencing. LN-α5 promoter methylation didn’t show significant difference between PE and normal placentas. Conclusion: LN-α5 downregulation is associated with PE placenta and impairs trophoblast viability and invasiveness, which could be a causative factor of PE pathogenesis.

An optimized strategy for cloning-based locus-specific bisulfite sequencing PCR

ABSTRACTIn this methods paper, we describe a successful strategy to investigate locus-specific methylation by cloning-based bisulfite sequencing. We cover sample handling, DNA isolation, DNA quality control before bisulfite conversion, bisulfite conversion, DNA quality control after bisulfite conversion, in silico identification of CpG islands, methylation-independent bisulfite sequencing PCR (BSP) assay design, methylation-independent BSP, cloning strategy, sequencing and data analysis. Methods that are described nicely elsewhere will not be covered in detail. Instead, the focus will be on tips/tricks and new methods/strategies used in this protocol, including quality control assessment of the DNA before and after bisulfite conversion and a pooled cloning strategy to reduce time, costs and effort during this step. In addition we comment on dealing with bias and improving overall protocol efficiency.

Hypermethylation of DLX4 predicts poor clinical outcome in patients with myelodysplastic syndrome

Background:Hypermethylation ofMethods:Real-time quantitative methylation-specific PCR and bisulfite sequencing PCR were carried out to detect the level ofResults:Conclusions:Our study indicated that