Aberrant Promoter Methylation Research Articles

Aberrant methylation of IGF2-AS promoter in early pregnancy loss

ObjectiveThe present study aimed to evaluate insulin-like growth factor 2 antisense (IGF2-AS) in the villi of human embryos and compared its expression between normal pregnancy and early pregnancy loss (EPL). Materials and methodsThe present study conducted a microarray analysis to identify the expression profiles of lncRNAs in villi from EPL and normal controls (controls, n = 10 and EPL patients, n = 10). Embryonic villi were collected from women who underwent artificial abortion. QPCR was used to confirm the results. The DNA methylation patterns were analyzed using pyrosequencing and bisulfite sequencing polymerase chain reaction. The percentage of methylation was compared in chorionic villi from the two groups. ResultsA total of 57 deregulated differentially expressed lncRNAs were detected, of which 33 were upregulated, and 24 were downregulated. The expression of lncRNA IGF2-AS was downregulated significantly in EPL villi compared with the normal villi. Negative regulation of IGF2-AS may be involved in the development of EPL. Methprimer predicted that IGF2-AS promoter had CpG islands and dense CG sites. Increased methylation at CpG islands present in IGF2-AS gene promoter was observed in EPL villi. ConclusionAn increase in methylation of IGF2-AS likely leads to its downregulation in chorionic villi of EPL. The findings suggest that a deficiency of IGF2-AS in the villi is associated with human EPL.

Aberrant SOCS3 Promoter Methylation as a Noninvasive Diagnostic Biomarker for Locally Advanced Prostate Cancer.

Objective:The aim of this study was to investigate the promoter methylation status of Rasassociated domain family 1A (RASSF1A), O-6-methylguanine-DNA methyltransferase (MGMT), Phosphatase with tensin homology (PTEN) and Suppressor of cytokine signaling 3 (SOCS3) tumor suppressor genes and evaluate the clinical utility of these genes as noninvasive, blood-based epigenetic biomarkers for the diagnosis of Prostate Cancer (PCa).Method:A total of 41 consecutive patients and 10 healthy control groups were enrolled in the study. Pyrosequencing was performed to analyze the methylation levels of the promoter regions of the four tumor suppressor genes in patients compared to healthy controls.Results:The promoter methylation levels of RASSF1A, MGMT, PTEN and SOCS3 did not differ between the patient and control groups. However, SOCS3 promoter methylation level was significantly higher for patients having locally advanced PCa compared to those having localizedPCa (p<0.05).ConclusionOur results indicated that SOCS3 could be a useful, noninvasive blood-based epigenetic biomarker for the diagnosis of locally advanced PCa.

Epigenome-wide analysis of common warts reveals aberrant promoter methylation.

Epigenetic alteration of host DNA is a common occurrence in both low- and high-risk human papillomavirus (HPV) infection. Although changes in promoter methylation have been widely studied in HPV-associated cancers, they have not been the subject of much investigation in HPV-induced warts, which are a temporary manifestation of HPV infection. The present study sought to examine the differences in promoter methylation between warts and normal skin. To achieve this, DNA was extracted from 24 paired wart and normal skin samples and inputted into the Infinium MethylationEPIC BeadChip microarray. Differential methylation analysis revealed a clear pattern of hyper- and hypomethylation in warts compared to normal skin, and the most differentially methylated promoters were found within the EIF3EP2, CYSLTR1, C10orf99, KRT6B, LAMA4, and H3F3B genes as well as the C9orf30 pseudogene. Moreover, pathway analysis showed that the H3F3A, CDKN1A, and MAPK13 genes were the most common regulators among the most differentially methylated promoters. Since the tissue samples were excised from active warts, however, this differential methylation could either be a cellular response to HPV infection or an HPV-driven process to establish the wart and/or promote disease progression. Conclusively, it is apparent that HPV infection alters the methylation status of certain genes to possibly initiate the formation of a wart and maintain its presence.

Aberrant promoter methylation may be responsible for the control of CD146 (MCAM) gene expression during breast cancer progression.

The CD146 (also known as MCAM, MUC-18, Mel-CAM) was initially reported in 1987, as a protein crucial for the invasiveness of malignant melanoma. Recently, it has been confirmed that CD146 has been involved in progression and poor overall survival of many cancers including breast cancer. Importantly, in independent studies, CD146 was reported to be a trigger of epithelial to mesenchymal transition in breast cancer cells. The goal of our current study was to verify the potential involvement of epigenetic mechanism behind the regulation of CD146 expression in breast cancer cells, as it has been previously reported in prostate cancer. First, we analysed the response of breast cancer cell lines, differing in the initial CD146 mRNA and protein content, to epigenetic modifier, 5-aza-2-deoxycytidine, and subsequently the methylation status of CD146 gene promoter was investigated, using direct bisulfite sequencing. We observed that treatment with demethylating agent led to induction of CD146 expression in all analysed breast cancer cell lines, both at mRNA and protein level, what was accompanied by increased expression of selected mesenchymal markers. Importantly, CD146 gene promoter analysis showed aberrant CpG island methylation in 2 out of 3 studied breast cancer cells lines, indicating epigenetic regulation of CD146 gene expression. In conclusion, our study revealed, for the first time, that aberrant methylation maybe involved in expression control of CD146, a very potent EMT inducer in breast cancer cells. Altogether, the data obtained may provide the basis for novel therapies as well as diagnostic approaches enabling sensitive and very accurate detection of breast cancer cells.

Early detection of the major male cancer types in blood-based liquid biopsies using a DNA methylation panel

BackgroundLung (LC), prostate (PCa) and colorectal (CRC) cancers are the most incident in males worldwide. Despite recent advances, optimal population-based cancer screening methods remain an unmet need. Due to its early onset, cancer specificity and accessibility in body fluids, aberrant DNA promoter methylation might be a valuable minimally invasive tool for early cancer detection. Herein, we aimed to develop a minimally invasive methylation-based test for simultaneous early detection of LC, PCa and CRC in males, using liquid biopsies.ResultsCirculating cell-free DNA was extracted from 102 LC, 121 PCa and 100 CRC patients and 136 asymptomatic donors’ plasma samples. Sodium-bisulfite modification and whole-genome amplification was performed. Promoter methylation levels of APCme, FOXA1me, GSTP1me, HOXD3me, RARβ2me, RASSF1Ame, SEPT9me and SOX17me were assessed by multiplex quantitative methylation-specific PCR.SEPT9me and SOX17me were the only biomarkers shared by all three cancer types, although they detected CRC with limited sensitivity. A “PanCancer” panel (FOXA1me, RARβ2me and RASSF1Ame) detected LC and PCa with 64% sensitivity and 70% specificity, complemented with “CancerType” panel (GSTP1me and SOX17me) which discriminated between LC and PCa with 93% specificity, but with modest sensitivity. Moreover, a HOXD3me and RASSF1Ame panel discriminated small cell lung carcinoma from non-small cell lung carcinoma with 75% sensitivity, 88% specificity, 6.5 LR+ and 0.28 LR–. An APCme and RASSF1Ame panel independently predicted disease-specific mortality in LC patients.ConclusionsWe concluded that a DNA methylation-based test in liquid biopsies might enable minimally invasive screening of LC and PCa, improving patient compliance and reducing healthcare costs. Moreover, it might assist in LC subtyping and prognostication.

Dickkopf Homolog 3 (DKK3) Acts as a Potential Tumor Suppressor in Gallbladder Cancer.

Gallbladder cancer (GBC) is a common malignancy of biliary tract cancers and its incidence has been rising rapidly worldwide. The prognosis for this disease is dismal as most of the symptoms are non-specific leading to a definitive diagnosis only at a late stage. Loss of DKK3 gene is associated with a possible tumor suppressor role in human cancers. The role and regulation of DKK3 in GBC have not been studied. We found that DKK3 expression levels were low in GBC patients and cell lines. Treatment of GBC cell lines with demethylating agent 5-Aza- 2'-deoxycytidine enhances its expression, establishing impact of methylation on DKK3 expression. We observed low expression of DKK3 in gallbladder adenocarcinoma tumors and highly invasive GBC cell lines. We showed that overexpression of DKK3 can decrease cell invasion, proliferation, and colony forming ability of GBC cells. Our data thus demonstrated the DKK3 gene is a potential tumor suppressor gene in GBC and aberrant promoter methylation could be involved in its downregulation, which may play a role in the tumorigenesis and aggressiveness of GBC.

C-Myc Acts as a Competing Endogenous RNA to Sponge miR-34a, in the Upregulation of CD44, in Urothelial Carcinoma

MicroRNAs (miRNAs) have been shown to play a crucial role in the progression of human cancers, including urothelial carcinoma (UC), the sixth-most common cancer in the world. Among them, miR-34a has been implicated in the regulation of cancer stem cells (CSCs); however, its role in UC has yet to be fully elucidated. In this study, bioinformatics and experimental analysis confirmed that miR-34a targets CD44 (a CSC surface marker) and c-Myc (a well-known cell cycle regulator) in UC. We found that, surprisingly, most UC cell lines and patient samples did express miR-34a, although epigenetic silencing by promoter hypermethylation of miR-34a expression was observed only in UMUC3 cells, and a subset of patient samples. Importantly, overexpression of c-Myc, a frequently amplified oncogene in UC, was shown to upregulate CD44 expression through a competing endogenous RNA (ceRNA) mechanism, such that overexpression of the c-Myc 3′UTR upregulated CD44, and vice versa. Importantly, we observed a positive correlation between the expression of c-Myc and CD44 in clinical samples obtained from UC patients. Moreover, overexpression of a dominant-negative p53 mutant downregulated miR-34a, but upregulated c-Myc and CD44, in UC cell lines. Functionally, the ectopic expression of miR-34a was shown to significantly suppress CD44 expression, and subsequently, suppression of cell growth and invasion capability, while also reducing chemoresistance. In conclusion, it appears that aberrant promoter methylation, and c-Myc-mediated ceRNA mechanisms, may attenuate the function of miR-34a, in UC. The tumor suppressive role of miR-34a in controlling CSC phenotypes in UC deserves further investigation.

Residual methylation of tumor suppressor gene promoters, RASSF6 and RASSF10, as novel biomarkers for minimal residual disease detection in adult acute lymphoblastic leukemia

Aberrant promoter methylation of RASSF6 and RASSF10 occurs at a high frequency in acute lymphoblastic leukemia (ALL). Because of the complexity of the current minimal residual disease (MRD) detecting-methods, the DNA methylation status of the RASSF6 and RASSF10 genes could potentially become biomarkers for the assessment of MRD levels in ALL patients. The promoter methylation status of RASSF6 and RASSF10 was assessed by using methylation-specific PCR (MSP) in the DNA isolated from 280 peripheral blood (PB) samples taken at the time of diagnosis, day 14, 28, and from the subsequent 30-month follow-ups of 45 adult ALL patients. The relative methylation level obtained during the follow-ups by MSP was compared to the MRD results obtained by the amplification of IG/TCR clonal rearrangements using the allele-specific quantitative-PCR (ASO-PCR) assay. Frequently, RASSF6 was methylated in B-ALL, and RASSF10 was methylated in T-ALL. The applicability and accuracy of the assays were increased when these markers were combined (91.1% and 93.8%, respectively). When a cutoff was defined for the PCR-MRD level, results of the 30months of MRD detection showed a significant correlation between the PCR and MSP techniques (r = 0.848; p < 0.001). Due to the high applicability, the non-invasiveness, and promising prospect of longitudinal assessment, the DNA methylation status of the RASSF6 and RASSF10 genes could be potential biomarkers for the assessment of residual disease in PB of patients with ALL.

Parkin gene mutations are not common, but its epigenetic inactivation is a frequent event and predicts poor survival in advanced breast cancer patients

BackgroundProgression of breast cancer involves both genetic and epigenetic factors. Parkin gene has been identified as a tumor suppressor gene in the pathogenesis of various cancers. Nevertheless, the putative role of Parkin in breast cancer remains largely unknown. Therefore, we evaluated the regulation of Parkin through both genetic and epigenetic mechanisms in breast carcinoma.MethodA total of 156 breast carcinoma and their normal adjacent tissue samples were included for mutational analysis through SSCP, and sequencing. MS-PCR was employed for methylation study whereas Parkin protein expression was evaluated using immunohistochemistry and western blotting. For the survival analysis, Kaplan–Meier curve and Cox’s proportional hazard model were used.ResultsIn expression analysis, Parkin protein expression was found to be absent in 68% cases of breast cancer. We found that aberrant promoter methylation of Parkin gene is a frequent incident in breast cancer tumors and cell lines. Our MS-PCR result showed that Parkin promoter methylation has a significant role (p = 0.0001) in reducing the expression of Parkin protein. Consistently, expression of Parkin was rectified by treatment with 5-aza-2-deoxycytidine. We also found significant associations of both Parkin negative expression and Parkin promoter methylation with the clinical variables. Furthermore, we found a very low frequency (5.7%) of Parkin mutation with no clinical significance. In survival analysis, patients having Parkin methylation and Parkin loss had a worse outcome compared to those harboring none of these events.ConclusionOverall, these results suggested that promoter methylation-mediated loss of Parkin expression could be used as a prognostic marker for the survival of breast cancer.

Mutation, methylation and expression analysis of LIFR gene in Indian breast cancer patients

LIFR functions as a tumor suppressor and metastatic suppressor of breast cancer. The present study investigates the status of LIFR gene in Indian breast cancer patients. A total of 137 breast cancer tissue and 137 adjacent normal tissue which served as controls were analyzed for mutation by automated DNA sequencing, methylation through methylation-specific polymerase chain reaction and its corresponding expression at mRNA and protein level using real-time quantitative polymerase chain reaction and immunohistochemistry respectively in Indian breast cancer patients. All the molecular findings were statistically correlated with clinopathological parameters of the patients to identify its association. LIFR mRNA expression was found to be 2.534 ± 3.52 fold downregulated with subsequent absence of protein in 67.15% cases (92/137). The absence of LIFR protein coincided with 80.95% (85/105) methylated cases thereby showing a very strong correlation among the LIFR promoter methylation and LIFR protein expression (p = 0.0001). We also observed G2968C nucleotide change in 6/137 cases of exon 20 of LIFR gene resulting in Glu990Gln mutation. Correlation of LIFR promoter methylation with geographic location and age at menopause and LIFR mutation with age at menarche, age at first live birth, molecular subtypes of breast cancer, and lymph node status remained significant even after bonferroni correction (p ≤ 0.0027). All these data suggests the relevance of these associations in relation to Indian breast cancer patients. The loss of LIFR protein was frequently found in Indian breast cancer patients, and aberrant promoter methylation showed a significant correlation with its downregulation.

Telomerase activation in small intestinal neuroendocrine tumours is associated with aberrant TERT promoter methylation, but not hot-spot mutations

ABSTRACTTelomere maintenance is a critical requirement for enabling replicative immortality and tumour development. Here, telomerase expression and activity, telomere length (TL) and potential regulatory factors that can underlie telomerase machinery alterations in small intestinal neuroendocrine tumours (SI-NETs) were analyzed. Telomerase activity assessed by TRAP assay was increased in SI-NETs compared to normal ileum (P < 0.001). The telomerase reverse transcriptase gene (TERT) was over-expressed in SI-NETs vs. normal ileal samples (P = 0.01). Furthermore, relative TL assessed by qPCR was found shorter in tumours compared with normal ileum (P = 0.02) and in distant metastasis samples compared to primary tumours and local metastases (P= 0.02). TERT promoter hotspot mutations were not present and TERT copy number gain was only observed in 3/70 tumour samples. TERT or chromosome 18 copy number alterations were not associated with telomerase expression and activity or TL. However, hypermethylation of TERT promoter in Region B – in the proximity of the transcription start site – was inversely correlated with TERT expression and telomerase activity and positively correlated with TL. Global LINE1 methylation was positively correlated with TERT promoter Region B methylation and was inversely correlated with telomerase activity, TERT expression and the upstream Region A methylation. The results show that telomerase activation, TERT expression and shorter telomeres are commonly found in SI-NETs. Aberrant DNA methylation of TERT promoter and of LINE1 can be implicated in abnormal regulation of TERT in SI-NETs.

MicroRNA-486-3p functions as a tumor suppressor in oral cancer by targeting DDR1

BackgroundDiscoidin domain receptor-1 (DDR1) tyrosine kinase is highly expressed in a variety of human cancers and involved in various steps of tumorigenesis. However, the precise mechanisms underlying the abnormal expression of DDR1 in oral squamous cell carcinoma (OSCC) has not been well investigated.MethodsThe expression of DDR1 on OSCC patients was determine by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. Specific targeting by miRNAs was determined by software prediction, luciferase reporter assay, and correlation with target protein expression. The functions of miR-486-3p and DDR1 were accessed by MTT and Annexin V analyses using gain- and loss-of-function approaches. Chromatin immunoprecipitation (ChIP) and methylation specific PCR (MSP) were performed to explore the molecular mechanisms by arecoline treatment.ResultsHere, we reported that DDR1 was significantly upregulated in OSCC tissues and its levels were inversely correlated with miR-486-3p expression. The experimental results in vitro confirmed that miR-486-3p decreased DDR1 expression by targeting the 3′-UTR of DDR1 mRNA. Overexpression of miR-486-3p led to growth inhibition and apoptosis induction with a similar function by knockdown of DDR1. Aberrant methylation of ANK1 promoter was a highly prevalent in OSCC and contributes to oral carcinogenesis by epigenetic silencing of ANK1 and miR-486-3p. We found that miR-486-3p can be transcriptionally co-regulated with its host gene ANK1 through epigenetic repression. DNA methylation inhibitor treatment re-expressed ANK1 and miR-486-3p. Importantly, arecoline, a major betel nut alkaloid, recruited DNMT3B binding to ANK1 promoter for DNA methylation and then attenuated the expression of miR-486-3p in OSCC.ConclusionThis study was the first to demonstrate that betel nut alkaloid may recruit DNMT3B to regulate miR-486-3p/DDR1 axis in oral cancer andmiR-486-3p and DDR1 may serve as potential therapeutic targets of oral cancer.

Genetic and epigenetic alterations of SDH genes in patients with sporadic succinate dehydrogenase-deficient gastrointestinal stromal tumors.

This study aimed to investigate the association of SDH gene mutations and promoter methylation with succinate dehydrogenase-deficient gastrointestinal stromal tumors (SDH-deficient GISTs) and to further discuss the potential molecular mechanisms underlying SDHB expression loss in these tumors. First, a total of 26 patients with SDH-deficient GISTs were selected by identifying the loss of SDHB protein expression and wild-type for KIT and PDGFRa mutations. Then SDH gene mutations and promoter methylation were detected by DNA sequencing and methylation-specific polymerase chain reaction, respectively, and the clinical and pathological data of SDH-deficient GISTs patients were collected and analyzed accordingly. The results of genetic testing demonstrated that 38.46% (10/26) of these patients harbored mutations in SDHB, SDHC, and SDHD genes (3 cases with double mutations). Besides, aberrant promoter methylation of SDH genes was detected in 10 out of 26 cases (38.46%), including 8 cases in SDHA gene, 3 cases in SDHB gene, 1 case in both SDHA and SDHB genes. It is suggested that SDH gene mutations and promoter methylation may contribute to the loss of SDH protein expression in sporadic SDH-deficient GISTs. This study indicated that the genetic and epigenetic alterations of SDH genes may occur during tumor formation.

Clinical significance of aberrant DEUP1 promoter methylation in hepatocellular carcinoma.

Accumulating studies have shown that methylation of tumor suppressor genes plays an important role in tumorigenesis. Deuterosome assembly protein 1 (DEUP1) has been implicated as a suppressor gene in some tumors and promoter methylation led to silencing of its expression. However, the roles of DEUP1 promoter methylation and expression in hepatocellular carcinoma (HCC) are not clear. In the present study, the expression and methylation of the DEUP1 promoter in HCC was investigated and the correlations with HCC occurrence and development were explored. A total of 60 HCC tumor and adjacent non-tumor tissues were included in this study. Reverse transcription-polymerase chain reaction, bisulfite PCR sequencing, immunohistochemistry and western blotting were applied to detect the methylation status of the DEUP1 promoter and its expression, and to analyze their associations with clinicopathological data. The results showed that the mRNA and protein expression of DEUP1 in adjacent non-tumor tissues was significantly increased compared with in the HCC tissues. DEUP1 promoter methylation was detected in 46/60 (76.7%) tumor tissues and there was a negative correlation between promoter methylation and DEUP1 protein expression (P<0.05). Analysis of the clinicopathological data revealed that the mRNA and protein expression of DEUP1, and its promoter methylation status, was associated with tumor node metastasis stage and tumor differentiation. Taken together, the results of the present study suggested that methylation of the DEUP1 promoter maybe an important mechanism for gene inactivation and has a critical role in the occurrence and development of liver cancer.

Investigation of the effect of P14 promoter aberrant methylation on the biological function of human lung cancer cells.

BackgroundThis study was conducted to investigate the effect of P14 promoter aberrant methylation on the biological function of human lung adenocarcinoma cells.MethodsWe used nested methylation‐specific PCR (NMSP) to detect the methylation status of the p14ARF promoter region in SPCA1 and BEAS2B cell lines. The experimental groups were treated with 5‐aza‐2′‐deoxycytidine (5‐Aza). Quantitative real‐time PCR, Western blot, flow cytometry, and Cell Counting Kit 8 were used to detect the expression of p14ARF messenger RNA and protein in each group, apoptosis, and cell proliferation inhibition, respectively.ResultsNMSP detected that the p14 promoter region of SPCA1 cells has abnormal methylation status. After treatment with 5‐Aza, the expression of p14ARF messenger RNA and protein in SPCA1 cells (P < 0.05) and the inhibition rate of cell proliferation (P < 0.05) were significantly increased, while the apoptosis rate was markedly increased (P < 0.05). However, no differences were observed in BEAS2B cells (P > 0.05).ConclusionAbnormal methylation of the p14ARF promoter region plays an important role in the development of lung cancer cells. Our results suggest the use of P14 promoter aberrant methylation as a therapeutic target for drug research or to improve the sensitivity of other drugs.

SHOX2 and WT1 Promoter Methylation Correlates with the Lung Cancer in Iranian Patients

Lung cancer is among the most common cause of cancer death in the world. Since the disease is diagnosed in the middle or late stages of the cancer development a more efficient method for an early diagnosis of the disease is required. The main goal of this study was to investigate the correlation between aberrant promoter methylation of the two genes: SHOX2(Short stature homeobox 2) and WT1(Wilms’ tumor gene1), in the plasma samples of the Iranian lung cancer patients. Following filling questionnaires and obtaining the consent of all participants, 70 cases including 45 patients and 25 healthy controls who underwent bronchoscopy procedure in the Masih-Daneshvari Hospital in Tehran, the blood samples were collected. DNA extraction was done and treatment was performed by sodium bisulfite applying Qiagen DNA methylation kit and Methylation specific PCR(MSP) was performed. The results showed a frequency of 82.2% and 55.55% of the hypermethylation for the SHOX2 and WT1 gene promoters in the caces samples, respectively. Our study demonstrated that hypermethylation of SHOX2 and WT1 genes promoter regions might be involved in lung cancer. The methylation of SHOX2 and WT1 in the blood plasma could be considered as the competent biomarkers for an early diagnosis of the lung cancer as a noninvasive method.

Genome-wide methylation profiling identified novel differentially hypermethylated biomarker MPPED2 in colorectal cancer

BackgroundEpigenetic alternation is a common contributing factor to neoplastic transformation. Although previous studies have reported a cluster of aberrant promoter methylation changes associated with silencing of tumor suppressor genes, little is known concerning their sequential DNA methylation changes during the carcinogenetic process. The aim of the present study was to address a genome-wide search for identifying potentially important methylated changes and investigate the onset and pattern of methylation changes during the progression of colorectal neoplasia.MethodsA three-phase design was employed in this study. In the screening phase, DNA methylation profile of 12 pairs of colorectal cancer (CRC) and adjacent normal tissues was analyzed by using the Illumina MethylationEPIC BeadChip. Significant CpG sites were selected based on a cross-validation analysis from The Cancer Genome Atlas (TCGA) database. Methylation levels of candidate CpGs were assessed using pyrosequencing in the training dataset (tumor lesions and adjacent normal tissues from 46 CRCs) and the validation dataset (tumor lesions and paired normal tissues from 13 hyperplastic polyps, 129 adenomas, and 256 CRCs). A linear mixed-effects model was used to examine the incremental changes of DNA methylation during the progression of colorectal neoplasia.ResultsThe comparisons between normal and tumor samples in the screening phase revealed an extensive CRC-specific methylomic pattern with 174,006 (21%) methylated CpG sites, of which 22,232 (13%) were hyermethylated and 151,774 (87%) were hypomethylated. Hypermethylation mostly occurred in CpG islands with an overlap of gene promoters, while hypomethylation tended to be mapped far away from functional regions. Further cross validation analysis from TCGA dataset confirmed 265 hypermethylated promoters coupling with downregulated gene expression. Among which, hypermethylated changes in MEEPD2 promoter was successfully replicated in both training and validation phase. Significant hypermethylation appeared since precursor lesions with an extensive modification in CRCs. The linear mixed-effects modeling analysis found that a cumulative pattern of MPPED2 methylation changes from normal mucosa to hyperplastic polyp to adenoma, and to carcinoma (P < 0.001).ConclusionsOur findings indicate that epigenetic alterations of MPPED2 promoter region appear sequentially during the colorectal neoplastic progression. It might be able to serve as a promising biomarker for early diagnosis and stage surveillance of colorectal tumorigenesis.

Differential regulation of CpG island methylation within divergent and unidirectional promoters in colorectal cancer.

The silencing of tumor suppressor genes by promoter CpG island (CGI) methylation is an important cause of oncogenesis. Silencing of MLH1 and BRCA1, two examples of oncogenic events, results from promoter CGI methylation. Interestingly, both MLH1 and BRCA1 have a divergent promoter, from which another gene on the opposite strand is also transcribed. Although studies have shown that divergent transcription is an important factor in transcriptional regulation, little is known about its implication in aberrant promoter methylation in cancer. In this study, we analyzed the methylation status of CGI in divergent promoters using a recently enriched transcriptome database. We measured the extent of CGI methylation in 119 colorectal cancer (CRC) clinical samples (65 microsatellite instability high [MSI‐H] CRC with CGI methylator phenotype, 28 MSI‐H CRC without CGI methylator phenotype and 26 microsatellite stable CRC) and 21 normal colorectal tissues using Infinium MethylationEPIC BeadChip. We found that CGI within divergent promoters are less frequently methylated than CGI within unidirectional promoters in normal cells. In the genome of CRC cells, CGI within unidirectional promoters are more vulnerable to aberrant methylation than CGI within divergent promoters. In addition, we identified three DNA sequence motifs that correlate with methylated CGI. We also showed that methylated CGI are associated with genes whose expression is low in normal cells. Thus, we here provide fundamental observations regarding the methylation of divergent promoters that are essential for the understanding of carcinogenesis and development of cancer prevention strategies.

A Preliminary Study of the Association between Apolipoprotein E Promoter Methylation and Atherosclerotic Cerebral Infarction

Aim: To investigate association of Apolipoprotein E (ApoE) gene promoter methylation with atherosclerotic cerebral infarction (ACI) in the Han Chinese population. Methods: Twenty-six ACI patients (the case group) and 26 healthy (the control group) were recruited randomly from Henan Han nationality population, from April 2016 to August 2016. Bisulfite pyrosequencing technology was used to examine the role of the aberrant gene promoter methylation in ACI in Han Chinese population. Especially, we used the odds ratio and 95% confidence interval (95% CI) method to elevate the correlation between ApoE Promoter Methylation and ACI. Results: The case group was significantly more likely to have hypertension and carotid atherosclerotic plaques (Table 1). The case group had significantly lower levels of high-density lipoprotein cholesterol (HDL-C), folate, and higher levels of homocysteine (Table 2). We observed that ACI patients (n = 26) had significantly higher methylation levels at cytosine-phosphate-guanine (CpG) 14 and CpG16 compared with controls (n = 26) (Table 3). Importantly, our results indicated a significant association between ApoE promoter methylation and ACI (OR = 16.146; 95% CI, 1.154-225.832; P* < .05; P*: adjusted for age, gender, carotid atherosclerotic plaque, hypertension, HDL-C, homocysteine, and folate) (Table 4). Conclusions: Our study indicates that ApoE promoter methylation may be associated with ACI in Han Chinese people.

SA47GENETIC PREDISPOSITION TO VITAMIN B12 AND FOLATE DEFICIENCY LEADS TO THE DEVELOPMENT OF DEPRESSION IN POSTPARTUM PERIOD BY HYPO-METHYLATION OF SEROTONIN TRANSPORTER AND MONOAMINE OXIDASE-A GENE LOCI

Plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) play a crucial role in cancer progression. In the present study we examined the regulation of PAI-1 and uPA expressions in normal prostate epithelial cells (PrEC) and the prostate cancer cell lines LNCaP, DU-145, and PC-3. The antigen and mRNA levels of PAI-1 were down-regulated in cancer cells, especially in LNCaP and DU-145. In the presence of proinflammatory cytokines, an increase of PAI-1 mRNA levels was observed in PrEC, LNCaP and PC-3, but not in DU-145 cells. Treatment with demethylating agent, 5-aza-2′-deoxycytidine increased the level of PAI-1 transcript in DU-145 cells and restored the inducing effect of cytokines on PAI-1 expression. An aberrant methylation of PAI-1 promoter in DU-145 and LNCaP cells was shown by methylation-sensitive high resolution melting (MS-HRM) analysis. PAI-1 methylation was also significantly increased in tumor samples (23.2 ± 1.7%) in comparison to adjacent non-tumor tissue (6.0 ± 0.8%). Furthermore, the expression of uPA was increased in high invasive cell lines DU-145 and PC-3 in comparison to PrEC and low invasive LNCaP cells. MS-HRM analysis revealed aberrant methylation of uPA promoter in LNCaP cells, but not in PrEC, DU-145 and PC-3 cells, as well as in normal and prostate cancer tissue samples. In conclusion, the study shows that PAI-1 and uPA expressions were changed in opposite directions in high invasive prostate cancer cell lines resulting in a strong decrease of PAI-1/uPA ratio, which may indicate a shift towards proteolytic activities. Methylation of the PAI-1 gene is suggested as one of the molecular mechanisms involved in the cancer-associated down-regulation of the PAI-1 expression.