Methylation-specific PCR Assay Research Articles

The expression of SOCS1 is regulated by promoter DNA methylation and is associated with mitochondria-mediated apoptosis of T-2 induced chondrocytes

At present, the function of SOCS1 in Kashin-Beck disease (KBD) has not been reported. This study aims to explore the expression and mechanism of SOCS1 in KBD, and provide theoretical basis for the prevention and treatment of KBD. The expression of SOCS1 were measured by qRT-PCR and Western blot. ELISA was used to detect the content of SOCS1 in serum and synovial fluid. CCK-8 kits were selected to measure the cell viability. Methylation Specific PCR (MSP) assay is used to detect the methylation level of SOCS1 in chondrocytes. Flow cytometry was used to analyze the apoptosis rate of chondrocytes in different groups. The expression of apoptosis related proteins (caspase-3 and caspase-9) and Cytochrome c were detected using Western blot. The mitochondrial ROS, ATP and the activity of mitochondrial respiratory chain complexes were detected using commercial kits. The results showed that the expression of SOCS1 significantly increases in KBD patients and T-2 induced chondrocytes. Further research has found that the methylation levels of SOCS1 were significantly reduced in KBD patients and T-2 induced chondrocytes. Functional studies have found that SOCS1 silencing inhibited chondrocyte apoptosis and mitochondrial dysfunction. More importantly, SOCS1 regulated mitochondrial mediated chondrocyte apoptosis through the IGF-1/IGF-1R/FAK/Drp1 pathway. In conclusion, SOCS1 expression is increased and methylation levels are decreased in KBD, and is involved in regulating mitochondrial mediated apoptosis in T-2 induced chondrocytes through IGF-1/IGF-1R/FAK/Drp1 signaling. This study provides new theoretical basis for the treatment and prevention of KBD in clinical practice.

CDKN2A promoter methylation enhances self-renewal of glioblastoma stem cells and confers resistance to carmustine

BackgroundGlioblastoma, a highly aggressive form of brain cancer, poses significant challenges due to its resistance to therapy and high recurrence rates. This study aimed to investigate the expression and functional implications of CDKN2A, a key tumor suppressor gene, in glioblastoma cells, building upon the existing background of knowledge in this field.MethodQuantitative reverse transcription PCR (qRT-PCR) analysis was performed to evaluate CDKN2A expression in U87 glioblastoma cells compared to normal human astrocytes (NHA). CDKN2A expression levels were manipulated using small interfering RNA (siRNA) and CDKN2A overexpression vector. Cell viability assays and carmustine sensitivity tests were conducted to assess the impact of CDKN2A modulation on glioblastoma cell viability and drug response. Sphere formation assays and western blot analysis were performed to investigate the role of CDKN2A in glioblastoma stem cell (GSC) self-renewal and pluripotency marker expression. Additionally, methylation-specific PCR (MSP) assays and demethylation treatment were employed to elucidate the mechanism of CDKN2A downregulation in U87 cells.ResultCDKN2A expression was significantly reduced in glioblastoma cells compared to NHA. CDKN2A overexpression resulted in decreased cell viability and enhanced sensitivity to carmustine treatment. CDKN2A inhibition promoted self-renewal capacity and increased pluripotency marker expression in U87 cells. CDKN2A upregulation led to elevated protein levels of p16INK4a, p14ARF, P53, and P21, which are involved in cell cycle regulation. CDKN2A downregulation in U87 cells was associated with high promoter methylation, which was reversed by treatment with a demethylating agent.ConclusionOur findings demonstrate that CDKN2A downregulation in glioblastoma cells is associated with decreased cell viability, enhanced drug resistance, increased self-renewal capacity, and altered expression of pluripotency markers. The observed CDKN2A expression changes are mediated by promoter methylation. These results highlight the potential role of CDKN2A as a therapeutic target and prognostic marker in glioblastoma.

CENPA knockdown restrains cell progression and tumor growth in breast cancer by reducing PLA2R1 promoter methylation and modulating PLA2R1/HHEX axis.

Breast cancer is a lethal malignancy affecting females worldwide. It has been reported that upregulated centromere protein A (CENPA) expression might indicate unfortunate prognosis and can function as a prognostic biomarker in breast cancer. This study aimed to investigate the accurate roles and downstream mechanisms of CENPA in breast cancer progression. CENPA protein levels in breast cancer tissues and cell lines were analyzed by Western blot and immunohistochemistry assays. We used gain/loss-of-function experiments to determine the potential effects of CENPA and phospholipase A2 receptor (PLA2R1) on breast cancer cell proliferation, migration, and apoptosis. Co-IP assay was employed to validate the possible interaction between CENPA and DNA methyltransferase 1 (DNMT1), as well as PLA2R1 and hematopoietically expressed homeobox (HHEX). PLA2R1 promoter methylation was determined using methylation-specific PCR assay. The biological capabilities of CENPA/PLA2R1/HHEX axis in breast cancer cells was determined by rescue experiments. In addition, CENPA-silenced MCF-7 cells were injected into mice, followed by measurement of tumor growth. CENPA level was prominently elevated in breast cancer tissues and cell lines. Interestingly, CENPA knockdown and PLA2R1 overexpression both restrained breast cancer cell proliferation and migration, and enhanced apoptosis. On the contrary, CENPA overexpression displayed the opposite results. Moreover, CENPA reduced PLA2R1 expression through promoting DNMT1-mediated PLA2R1 promoter methylation. PLA2R1 overexpression could effectively abrogate CENPA overexpression-mediated augment of breast cancer cell progression. Furthermore, PLA2R1 interacted with HHEX and promoted HHEX expression. PLA2R1 knockdown increased the rate of breast cancer cell proliferation and migration but restrained apoptosis, which was abrogated by HHEX overexpression. In addition, CENPA silencing suppressed tumor growth in vivo. CENPA knockdown restrained breast cancer cell proliferation and migration and attenuated tumor growth in vivo through reducing PLA2R1 promoter methylation and increasing PLA2R1 and HHEX expression. We may provide a promising prognostic biomarker and novel therapeutic target for breast cancer.

Cholesterol homeostasis confers glioma malignancy triggered by hnRNPA2B1-dependent regulation of SREBP2 and LDLR.

Dysregulation of cholesterol metabolism is a significant characteristic of glioma, yet the underlying mechanisms are largely unknown. N6-methyladenosine (m6A) modification has been implicated in promoting tumor development and progression. The aim of this study was to determine the key m6A regulatory proteins involved in the progression of glioma, which is potentially associated with the reprogramming of cholesterol homeostasis. Bioinformatics analysis was performed to determine the association of m6A modification with glioma malignancy from The Cancer Genome Atlas and Genotype-Tissue Expression datasets. Glioma stem cell (GSC) self-renewal was determined by tumor sphere formation and bioluminescence image assay. RNA sequencing and lipidomic analysis were performed for cholesterol homeostasis analysis. RNA immunoprecipitation and luciferase reporter assay were performed to determine hnRNPA2B1-dependent regulation of sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR) mRNA. The methylation status of hnRNPA2B1 promoter was determined by bioinformatic analysis and methylation-specific PCR assay. Among the m6A-regulatory proteins, hnRNPA2B1 was demonstrated the most important independent prognostic risk factor for glioma. hnRNPA2B1 ablation exhibited a significant tumor-suppressive effect on glioma cell proliferation, GSC self-renewal and tumorigenesis. hnRNPA2B1 triggers de novo cholesterol synthesis by inducing HMGCR through the stabilization of SREBP2 mRNA. m6A modification of SREBP2 or LDLR mRNA is required for hnRNPA2B1-mediated mRNA stability. The hypomethylation of cg21815882 site on hnRNPA2B1 promoter confers elevated expression of hnRNPA2B1 in glioma tissues. The combination of targeting hnRNPA2B1 and cholesterol metabolism exhibited remarkable antitumor effects, suggesting valuable clinical implications for glioma treatment. hnRNPA2B1 facilitates cholesterol uptake and de novo synthesis, thereby contributing to glioma stemness and malignancy.

MiR-488-3p facilitates wound healing through CYP1B1-mediated Wnt/β-catenin signaling pathway by targeting MeCP2.

Diabetic wounds are difficult to heal, but the pathogenesis is unknown. MicroRNAs (miRNAs) are thought to play important roles in wound healing. The effect of miR-488-3p in wound healing was studied in this article. The gene methylation was measured by methylation specific PCR (MSP) assay. A dual-luciferase reporter assay was adopted to analyze the interaction between miR-488-3p and MeCP2. Cytochrome P450 1B1 (CYP1B1) is a monooxygenase belonging to the cytochrome P450 family that aids in wound healing. Our findings showed that the miR-488-3p and CYP1B1 expression levels were much lower in wound tissues of diabetics with skin defects, but the methyl-CpG-binding protein 2 (MeCP2) level was significantly higher than that in control skin tissues. MiR-488-3p overexpression increased cell proliferation and migration, as well as HUVEC angiogenesis, while inhibiting apoptosis, according to function experiments. In vitro, MeCP2 inhibited wound healing by acting as a target of miR-488-3p. We later discovered that MeCP2 inhibited CYP1B1 expression by enhancing its methylation state. In addition, CYP1B1 knockdown inhibited wound healing. Furthermore, MeCP2 overexpression abolished the promoting effect of miR-488-3p on wound healing. It also turned out that CYP1B1 promoted wound healing by activating the Wnt4/β-catenin pathway. Animal experiments also showed that miR-488-3p overexpression could accelerate wound healing in diabetic male SD rats. MiR-488-3p is a potential therapeutic target for diabetic wound healing since it improved wound healing by activating the CYP1B1-mediated Wnt4/-catenin signaling cascade via MeCP2.

PD-L1 (CD274) promoter hypomethylation predicts immunotherapy response in metastatic urothelial carcinoma

ABSTRACT PD-L1 status assessed by immunohistochemistry (IHC) has failed to reliably predict outcomes for patients with metastatic urothelial carcinoma (mUC) on immune checkpoint blockade (ICB). PD-L1 promoter methylation is an epigenetic mechanism that has been shown to regulate PD-L1 mRNA expression in various malignancies. The aim of our present study was to evaluate the predictive potential of PD-L1 promoter methylation status (mPD-L1) in ICB-treated mUC compared to conventional IHC-based PD-L1 assessment. We quantified mPD-L1 in formalin-fixed and paraffin-embedded tissue sections using an established quantitative methylation-specific PCR assay (qMSP) in a well-characterized multicenter ICB-treated cohort comprising N = 107 patients with mUC. Additionally, PD-L1 protein expression in tumor tissues was assessed using regulatory approved IHC protocols. The effect of pharmacological hypomethylation by the DNA methyltransferase inhibitor decitabine in combination with interferon-γ stimulation in urothelial carcinoma cell lines was investigated by IHC and FACS. mPD-L1 hypomethylation predicted objective response rate at the first staging on ICB. Patients with tumors categorized as PD-L1 hypomethylated (lower quartile) showed significantly prolonged progression-free (PFS) and overall survival (OS) after ICB initiation. In contrast, PD-L1 protein expression status neither correlated with response nor survival. In multivariable Cox regression analyses, PD-L1 promoter hypermethylation remained an independent predictor of unfavorable PFS and OS. In urothelial carcinoma cell lines, pharmacological demethylation led to an upregulation of membranous PD-L1 expression and an enhanced inducibility of PD-L1 expression by interferon γ. Hypomethylation of the PD-L1 promoter is a promising predictive biomarker for response to ICB in patients with mUC.

B-382 Accurate and Early Detection of Colorectal Cancer Using a Multilocus DNA Methylation Markers-based Testing in Peripheral Blood Mononuclear Cells

Abstract Background An effective blood-based method for the diagnosis of colorectal cancer (CRC) has not yet been developed. Molecular alterations of immune cells occur early in tumorigenesis, providing the theoretical underpinning for early cancer diagnosis. Therefore, we aimed to develop an effective multi-molecular simultaneous detection method based on peripheral blood mononuclear cells (PBMCs) to improve the diagnosis of early-stage CRC. Methods Candidate DNA methylation markers were first identified from PBMCs using the Infinium MethylationEPIC array in the discovery phase (Shandong cohort I, n = 100) and further validated via pyrosequencing and targeted bisulfite sequencing in validation phase (Shandong cohort II, n = 202). Then, a single-tube multiplex methylation-specific quantitative PCR assay (multi-msqPCR) for simultaneous detection of five markers was established in a pilot study. After that, a CRC prediction model (CPM) based on DNA methylation and multi-msqPCR method was construct and its diagnostic performance were evaluated using the area under the receiver operating characteristic curve (AUROC) in our multicenter cohort (n = 595). Results Five discriminative DNA methylation markers identified by Illumina 850K microarray were successfully validated to diagnose early-stage CRC. Multi-msqPCR showed a better discriminative performance and 10-time lower detection limit than single-molecule detection in early-stage CRC. The cross-validated AUROC for CPM for early-stage CRC was 0.91 (sensitivity = 81.18%; specificity = 89.39%), significantly higher than CEA (AUROC = 0.62). CPM assay also yielded a high degree of discrimination for advanced adenoma (AA) cases (AUROC = 0.85; sensitivity = 63.04%; specificity = 89.39%). Besides, detecting CPM in multiple cancer types implied a CRC-specific diagnostic value. Our follow-up data also demonstrated that CPM could detect early-stage CRC up to 2 years before current traditional diagnostic methods. Conclusions CPM, in combination with epigenetic biomarkers and the multi-msqPCR method, was promising, cost-effective, and easily implementable for routine clinical diagnosis of early-stage CRC.

B-381 A Multiplex Blood-based Assay Targeting DNA methylation in Peripheral Blood Mononuclear Cells Enables Early Detection of Breast Cancer

Abstract Background The immune system can monitor the development of tumors, and the DNA methylation is involved in the body immune response to tumors. Therefore, the DNA from peripheral blood mononuclear cell (PBMC) that carries cancer-specific epigenetic aberrations may serve as a non-invasive marker for early detection of breast cancer (BC). Methods Genome-wide DNA methylation profiling on PBMC DNA from BC patients and healthy controls (Cohort I, n = 80) was performed in the discovery phase by Infinium 850K BeadChips. The candidate CpG sites were selected and validated in a multistep validation phase (Cohort II, n = 200) via pyrosequencing and targeted bisulfite sequencing. After that, the multiplex quantitative methylation-specific PCR assay (mBC-MSP) was established to detect BC, and the performance of this diagnostic method was analyzed in a multicenter cohort (Cohort III, n = 501). Results A total of 289 differentially methylated CpG positions (DMPs) between BC patients and healthy controls were identified. Among them, eight significant DMPs markers were selected and further validated using pyrosequencing and targeted bisulfite sequencing, and four hyper-methylated DMPs (cg18637238, cg16652347, cg13828440, cg11754974) passed the final validation. Then, we developed a mBC-MSP assay based on the four methylated markers. This assay exhibited excellent performance in distinguishing BC patients, with an AUC of 0.925, sensitivity of 83.1%, and specificity of 90.4%. More importantly, compared with CA153, CA125 and CEA, the mBC-MSP assay had higher sensitivity for early-stage BC (stage 0, 88.2% vs 0%, 0%, 5.9%) and minimal tumor with diameter ≤1.5 cm (91.7% vs 0%, 2.1%, 0%). Conclusions The mBC-MSP assay based on the four identified DNA methylation markers from PBMC can serve as a noninvasive, accurate, rapid, and high-throughput method for early diagnosis of BC, and it is more sensitive than traditional tumor markers for detecting early-stage BC and minimal tumor.

OCLN as a novel biomarker for prognosis and immune infiltrates in kidney renal clear cell carcinoma: an integrative computational and experimental characterization.

Occludin (OCLN) is an important tight junction protein and has been reported to be abnormally expressed in the development of malignant tumors. However, its biomarker and carcinogenic roles in kidney renal clear cell carcinoma (KIRC) are less investigated. The Cancer Genome Atlas database and Human Protein Atlas database were used to analyze the expression of OCLN in KIRC. UALCAN database and methylation-specific PCR assay were used to evaluate the methylation level of OCLN in KIRC. Univariate and multivariate Cox regression analyses were performed to model the prognostic significance of OCLN in KIRC patient cohorts. The correlation between OCLN expression and the immune cell infiltration, immune-related function and immune checkpoints were explored. Finally, EdU, scratch assay and transwell experiments were conducted to validate the role of OCLN in KIRC development. The expression of OCLN was significantly downregulated in KIRC, compared with normal renal tissues (p<0.001). Patients with low OCLN expression showed a worse prognosis and poorer clinicopathological characteristics. Functional enrichment analysis revealed that OCLN was mainly involved in biological processes such as immune response, immunoglobulin complex circulating and cytokine and chemokine receptor to mediate KIRC development. Immune-related analysis indicated that OCLN could potentially serve as a candidate target for KIRC immunotherapy. OCLN overexpression inhibited proliferation, migration and invasion of KIRC cells in vitro. OCLN was validated as a candidate prognostic biomarker and therapeutic target of KIRC based both on computational and experimental approaches. More in vivo experiments will be conducted to decode its molecular mechanism in KIRC carcinogenesis in the future work.

Evaluation of RASSF1A methylation in the lysate of sentinel lymph nodes for detecting breast cancer metastasis: A diagnostic accuracy study.

The restriction enzyme-based digital methylation-specific polymerase chain reaction (RE-dMSP) assay is useful for diagnosing sentinel lymph node (SN) metastasis in patients with breast cancer, by detecting tumor-derived methylated Ras association domain-containing protein 1 (RASSF1A). In addition, this assay has high concordance (95.0%) with one-step nucleic acid amplification (OSNA). The present study aimed to perform RE-dMSP using OSNA lysate from more patients and to re-evaluate its clinical usage. Overall, 418 SNs from 347 patients were evaluated using both OSNA and RE-dMSP. The concordance rate was 83.3% (348/418). RASSF1A methylation of the primary tumors was negative in 36 patients. When these patients were excluded, the concordance rate improved to 88.2% (330/374). Of the 79 OSNA-negative cases, 19 were RE-dMSP-positive, although all were positive for cytokeratin 19 expression in the primary tumor, suggesting that RE-dMSP can detect tumor-derived DNA with a higher sensitivity. The percent of methylated reference of the breast tumors showed a wide variety in the 16 OSNA-positive/RE-dMSP-negative cases, and such variability of methylation could have affected the results in these patients. In conclusion, although RE-dMSP can diagnose SN metastasis with high sensitivity and accuracy, and can be a supplementary tool to OSNA in breast cancer, RE-dMSP showed certain discordance with OSNA and critically depended on the absence or heterogeneity of DNA methylation in breast tumors. Further research is expected to develop an assay targeting other DNA alterations, such as mutations.

BRAP silencing protects against neuronal inflammation, oxidative stress and apoptosis in cerebral ischemia-reperfusion injury by promoting PON1 expression.

BRCA1 associated protein (BRAP) participates in the regulation of myocardial infarction and atherosclerosis. But the function of BRAP in cerebral ischemia-reperfusion (CIR) injury has not been elucidated yet. BRAP expression in PC12 cells in response to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment was examined with Western blot assay. PC12 cells underwent OGD/R-treatment and were subsequently transfected with pcDNA-BRAP or sh-BRAP, followed by determination of viability, lactate dehydrogenase (LDH) production, apoptosis, inflammatory cytokine secretion, and oxidative stress marker protein levels. Paraoxonase 1 (PON1) promoter methylation was evaluated with methylation-specific PCR assay. the effect of BRAP/PON1 axis on CIR injury was investigated by rescue experiments. Additionally, sh-BRAP was injected into a middle cerebral artery occlusion (MCAO) rat model, and the changes of neurological damage were evaluated. BRAP overexpression exacerbated OGD/R-induced viability reduction, LDH production, apoptosis, inflammatory cytokine secretion and oxidative stress in PC12 neuronal cells. In contrast, BRAP silencing showed the opposite results. Mechanistically, BRAP reduced PON1 expression by promoting DNA methyl transferase1 (DNMT1)-mediated PON1 promoter methylation. PON1 silencing reversed BRAP-mediated neuroprotection. Additionally, BRAP silencing alleviated CIR-induced neurological damage in MCAO rats. BRAP silencing suppressed OGD/R-induced neuronal apoptosis, inflammation, and oxidative stress, and alleviated CIR-induced neurological damage in MCAO rats through facilitating PON1 expression.

Abnormal Promoter Methylation of Nucleotide-Binding Oligomerization Domain Containing 2 (NOD2) Gene in the Pathogenesis of Crohn’s Disease

Changes in the expression of nucleotide-binding oligomerization domain containing 2 (NOD2) play an important role in the pathogenesis of several autoimmune diseases, such as inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC). It seems that epigenetic modifications, including DNA methylation, have an important role in the suppression of gene activity. In this study, the relationship between DNA methylation patterns of the promoter region of the NOD2 gene and the pathogenesis of CD was assessed. Colonic mucosal samples were obtained from 15 Iranian patients with CD and 15 matched healthy controls with no history of autoimmune diseases. After bisulfite conversion of genomic DNA, the DNA methylation status of three CpG sites in the promoter region of the NOD2 gene was determined by the real-time quantitative multiplex methylation-specific PCR assay. Using this approach, we identified a decreased level of methylation of the NOD2 promoter in the colonic mucosa of patients with CD (0.128±0.093 vs. 0.025±0.016, unmethylated DNA in CD vs. healthy controls, respectively, P&lt;0.000). According to our findings, promoter hypomethylation of the NOD2 gene in the colonic mucosa might contribute to the development and severity of CD.

DNA methylation-based testing in peripheral blood mononuclear cells enables accurate and early detection of colorectal cancer.

An effective blood-based method for the diagnosis of colorectal cancer (CRC) has not yet been developed. Molecular alterations of immune cells occur early in tumorigenesis, providing the theoretical underpinning for early cancer diagnosis based on immune cell profiling. Therefore, we aimed to develop an effective detection method based on peripheral blood mononuclear cells (PBMCs) to improve the diagnosis of CRC. Analysis of the genome-wide methylation landscape of PBMCs from CRC patients and healthy controls by microarray, pyrosequencing, and targeted bisulfite sequencing revealed five DNA methylation markers for CRC diagnosis, especially early-stage CRC (eCRC). A single-tube multiple methylation-specific quantitative PCR assay (multi-msqPCR) for simultaneous detection of five methylation markers was established, which allowed quantitative analysis of samples with as little as 0.1% PBMC DNA and had better discriminative performance than single-molecule detection. Then, a CRC diagnostic model (CDM) based on methylation markers and the multi-msqPCR method was constructed that achieved high accuracy for eCRC (AUC=0.91; sensitivity=81.18%; specificity=89.39%), which was improved compared to CEA (AUC=0.79). The CDM also enabled a high degree of discrimination for advanced adenoma (AA) cases (AUC=0.85, sensitivity=63.04%). Follow-up data also demonstrated that the CDM could identify CRC potential up to 2 years before currently used diagnostic methods. In conclusion, the approach constructed in this study based on PBMC-derived DNA methylation markers and a multi-msqPCR method is a promising and easily implementable diagnostic method for eCRC.

A multiplex blood-based assay targeting DNA methylation in PBMCs enables early detection of breast cancer

The immune system can monitor tumor development, and DNA methylation is involved in the body’s immune response to tumors. In this work, we investigate whether DNA methylation alterations in peripheral blood mononuclear cells (PBMCs) could be used as markers for early detection of breast cancer (BC) from the perspective of tumor immune alterations. We identify four BC-specific methylation markers by combining Infinium 850 K BeadChips, pyrosequencing and targeted bisulfite sequencing. Based on the four methylation markers in PBMCs of BC, we develop an efficient and convenient multiplex methylation-specific quantitative PCR assay for the detection of BC and validate its diagnostic performance in a multicenter cohort. This assay was able to distinguish early-stage BC patients from normal controls, with an AUC of 0.940, sensitivity of 93.2%, and specificity of 90.4%. More importantly, this assay outperformed existing clinical diagnostic methods, especially in the detection of early-stage and minimal tumors.

Elevated EVL Methylation Level in the Normal Colon Mucosa Is a Potential Risk Biomarker for Developing Recurrent Adenomas.

Individuals with adenomatous colorectal polyps undergo repeated colonoscopy surveillance to identify and remove metachronous adenomas. However, many patients with adenomas do not develop recurrent adenomas. Better methods to evaluate who benefits from increased surveillance are needed. We evaluated the use of altered EVL methylation as a potential biomarker for risk of recurrent adenomas. Patients with ≥1 colonoscopy had EVL methylation (mEVL) measured with an ultra-accurate methylation-specific droplet digital PCR assay on normal colon mucosa. The association between EVL methylation levels and adenoma or colorectal cancer was evaluated using three case/control definitions in three models: unadjusted (model 1), adjusting for baseline characteristics (model 2), and an adjusted model excluding patients with colorectal cancer at baseline (model 3). Between 2001 and 2020, 136 patients were included; 74 healthy patients and 62 patients with a history of colorectal cancer. Older age, never smoking, and baseline colorectal cancer were associated with higher levels of mEVL (P ≤ 0.05). Each log base 10 difference in mEVL was associated with an increased risk of adenoma(s) or cancer at/after baseline for model 1 [OR, 2.64; 95% confidence interval (CI), 1.09-6.36], and adenoma(s) or cancer after baseline for models 1 (OR, 2.01; 95% CI, 1.04-3.90) and model 2 (OR, 3.17; 95% CI, 1.30-7.72). Our results suggest that EVL methylation level detected in the normal colon mucosa has the potential to be a biomarker for monitoring the risk for recurrent adenomas. These findings support the potential utility of EVL methylation for improving the accuracy for assigning risk for recurrent colorectal adenomas and cancer.

Genotoxicity and hypomethylation of LINE-1 induced by electronic cigarettes

Currently, the marketing of electronic cigarettes as a safe alternative to smoking has increased, which is associated with greater use of these devices, especially among young people and smokers interested in quitting tobacco cigarettes. Given the growing use of this type of product, there is a need to determine the consequences of electronic cigarettes on human health, especially since many of the compounds contained in the aerosol and liquid of these devices have a high potential to be carcinogenic and genotoxic. Additionally, many of these compounds' aerosol concentrations exceed the safe limits. We have evaluated the levels of genotoxicity and changes in DNA methylation patterns associated with vaping. We analyzed a total of 90 peripheral blood samples from a population of vapers (n = 32), smokers (n = 18), and controls (n = 32), in which the frequencies of genotoxicity were determined by the cytokinesis-blocking micronuclei (CBMN) assay and the patterns of methylation of the repetitive elements of LINE-1 through the Quantitative Methylation Specific PCR (qMSP) assay. Here we show an increase in genotoxicity levels associated with vaping habits. Additionally, the group of vapers showed changes at the epigenetic level specifically associated with the loss of methylation of the LINE-1 elements. These changes in LINE-1 methylation patterns were reflected in its representative RNA expression detected in vapers.

Promoter hypermethylation analysis of host genes in cervical intraepithelial neoplasia and cervical cancers on histological cervical specimens

BackgroundDNA methylation is an essential factor in the progression of cervical intraepithelial neoplasia (CIN) to cervical cancer. The aim was to investigate the diagnostic value provided by methylation biomarkers of six tumor suppressor genes (ASTN1, DLX1, ITGA4, RXFP3, SOX17 and ZNF671) for cervical precancerous lesions and cervical cancer.MethodsThe histological cervical specimens of 396 cases including 93 CIN1, 99 CIN2, 93 CIN3 and 111 cervical cancers were tested for methylation-specific PCR assay (GynTect®) of score and positive rate. Among them, 66 CIN1, 93 CIN2, 87 CIN3 and 72 cervical cancers were further used for paired analysis. A chi-square test was used to analyze the difference of methylation score and positive rate in cervical specimens. The paired t-test and paired chi-square test were for analyzing the methylation score and positive rate in paired CIN and cervical cancer cases. The specificity, sensitivity, odds ratio (OR) and 95% confidence interval (95% CI) of the GynTect® assay for CIN2 or worse (CIN2 +) and CIN3 or worse (CIN3 +) were evaluated.ResultsAccording to the chi-square test trend, hypermethylation increased with severity of the lesions as defined by histological grading (P = 0.000). The methylation score above 1.1 was more common in CIN2 + than in CIN1. The DNA methylation scores in the paired groups of CIN1, CIN3 and cervical cancer were significant differences (P = 0.033, 0.000 and 0.000, respectively), except for CIN2 (P = 0.171). While the positive rate of GynTect® in each paired group had no difference (all P > 0.05). The positive rate of every methylation marker in the GynTect® assay showed differences in four cervical lesion groups (all P < 0.05). The specificity of GynTect® assay for detection of CIN2 + /CIN3 + were higher than high-risk human papillomavirus test. With CIN1 as a reference, the positive status of GynTect®/ZNF671 were significantly higher in CIN2 + : odds ratio (OR) 5.271/OR 13.909, and in CIN3 + : OR 11.022/OR 39.150, (all P < 0.001).ConclusionThe promoter methylation of six tumor suppressor genes is related to the severity of cervical lesions. The GynTect® assay based on cervical specimens provides diagnostic values for detecting CIN2 + and CIN3 + .

Runt-related transcription factor 3, mediated by DNA-methyltransferase 1, regulated Schwann cell proliferation and myelination during peripheral nerve regeneration via JAK/STAT signaling pathway

Schwann cells (SCs) play a crucial role in peripheral nerve injury and regeneration. Recently, RUNX3 was found to be linked with neurological dysfunction. We examined the RUNX3 expression in sciatic nerve stumps with peripheral nerve injury of rats, cyclic adenosine monophosphate (cAMP)-induced SCs. MTT assay was applied to determine the proliferation of SCs. Cell migration and apoptosis were assessed using wound healing assay and flow cytometry. Subsequently, we detected the methylation level of RUNX3 using Methylation-specific PCR assay and verified its regulation by DNMT1. The RUNX3 expressions were increased in sciatic nerve stumps with peripheral nerve injury and cAMP-induced SCs differentiation, which were related to demethylation of its promoter region regulated by DNMT1. RUNX3 knockdown notably suppressed the proliferation and migration, and induced the cell apoptosis of SCs. Silencing of RUNX3 inhibited the cAMP-induced morphological changes of SCs and the increase of myelin-related proteins induced by cAMP in SCs, while RUNX3 overexpression exerted opposite effects. Besides, the overexpression of RUNX3 promoted the activation of JAK/STAT signaling to regulate SCs proliferation and myelination. Meanwhile, DNMT1 overexpression inhibited the expression of RUNX3, and cell proliferation and myelination. In conclusion, RUNX3 mediated by DNMT1 regulated SC proliferation and myelination via JAK/STAT signaling pathway.

Mitophagy-promoting miR-138-5p promoter demethylation inhibits pyroptosis in sepsis-associated acute lung injury.

The present study was designed to explore the potential regulatory mechanism between mitophagy and pyroptosis during sepsis-associated acute lung injury (ALI). In vitro or in vivo models of sepsis-associated ALI were established by administering lipopolysaccharide (LPS) or performing caecal ligation and puncture (CLP) surgery. Pyroptosis levels were detected by electron microscopy, immunofluorescence, flow cytometry, western blotting and immunohistochemistry. Dual-luciferase reporter gene assay was applied to verify the targeting relationship between miR-138-5p and NLRP3. Methylation-specific PCR and chromatin immunoprecipitation assays were used to determine methylation of the miR-138-5p promoter. Mitophagy levels were examined by transmission electron microscopy and western blotting. NLRP3 inflammasome silencing alleviated alveolar macrophage (AM) pyroptosis and septic lung injury. In addition, we confirmed the direct targeting relationship between miR-138-5p and NLRP3. Overexpressed miR-138-5p alleviated AM pyroptosis and the pulmonary inflammatory response. Moreover, the decreased expression of miR-138-5p was confirmed to depend on promoter methylation, while inhibition of miR-138-5p promoter methylation attenuated AM pyroptosis and pulmonary inflammation. Here, we discovered that an increased cytoplasmic mtDNA content in sepsis-induced ALI models induced the methylation of the miR-138-5p promoter, thereby decreasing miR-138-5p expression, which may activate the NLRP3 inflammasome and trigger AM pyroptosis. Mitophagy, a form of selective autophagy that clears damaged mitochondria, reduced cytoplasmic mtDNA levels. Furthermore, enhanced mitophagy might suppress miR-138-5p promoter methylation and relieve the pulmonary inflammatory response, changes that were reversed by treatment with isolated mtDNA. In summary, our study indicated that mitophagy induced the demethylation of the miR-138-5p promoter, which may subsequently inhibit NLRP3 inflammasome, AM pyroptosis and inflammation in sepsis-induced lung injury. These findings may provide a promising therapeutic target for sepsis-associated ALI.

Long noncoding RNA HOTAIR facilitates pulmonary vascular endothelial cell apoptosis via DNMT1 mediated hypermethylation of Bcl-2 promoter in COPD

BackgroundTo study the regulatory effect of Long non-coding RNA (LncRNA) HOX transcript antisense RNA (HOTAIR) on pulmonary vascular endothelial cell (HPVEC) apoptosis and determine whether the HOTAIR facilitate HPVEC apoptosis via DNMT1 mediated hypermethylation of Bcl-2 promoter in chronic obstructive pulmonary disease (COPD).MethodsLncRNA array was used to measure the differentially expressed lncRNAs in COPD and non-COPD lung tissues. Expression of HOTAIR in COPD patient lungs and cigarette smoke extract (CSE)-induced HPVEC was assessed by qRT-PCR. The location of HOTAIR was determined in COPD patient lungs and HPVEC by RNA in situ hybridization (RNA-ISH). The emphysema mouse model and HOTAIR knockdown mice were each established by inhaling cigarette smoke or intratracheal lentiviral vectors instillation. The dysregulation of DNA methyltransferase enzyme 1 (DNMT1), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and Cleaved-caspase 3 protein expression were detected by Western blotting. HOTAIR, DNMT1, Bcl-2 and Bax mRNA expression were measured by quantitative real-time polymerase chain reaction. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays were used to assess apoptotic ratio in mice and CSE-induced HPVEC. Methylation-specific PCR (MSP) assay was conducted to observe the alterations in the methylation of the Bcl-2 promoter in specimens. RNA pull-down assay was used for analysis of the correlation between HOTAIR and DNMT1.ResultsThe expression levels of the HOTAIR were up-regulated in COPD patient lungs and CSE-induced HPVEC. HPVEC apoptosis with down-regulated Bcl-2 expression, increased promoter methylation, DNMT1, Bax and Cleaved-caspase 3 expression was found in emphysema mouse model and CSE-induced HPVEC. Knockdown HOTAIR can attenuate cell apoptosis and emphysema via DNMT1 mediated hypermethylation of Bcl-2 promoter in mice. In vitro, HOTAIR can aggravate the apoptosis of CSE-exposed HPVEC. DNMT1 was a target of HOTAIR and had a positive correlation with HOTAIR.ConclusionHOTAIR facilitates HPVEC apoptosis via DNMT1 mediated hypermethylation of Bcl-2 promoter in COPD, and attenuating the expression of HOTAIR may be a new therapy to prevent COPD.