Xiaoxia Ye

Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry

We developed and validated a rapid, sensitive, and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of global DNA methylation in tissue. DNA was extracted by phenol-chloroform, hydrolyzed using 88% formic acid at 140°C, spiked with cytosine-2,4-(13)C(15)N(2) as internal standard, evaporated under nitrogen, reconstituted in methanol, and analyzed by LC-MS/MS in multiple reaction monitoring mode to reflect the global DNA methylation of the tissue. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 0.5pg for both cytosine (Cyt) and 5-methylcytosine (5mCyt). The linear range of calibration curve was 1-50 and 1-100ng/ml for 5mCyt and Cyt, respectively, with a correlation coefficient higher than 0.99. The relative standard deviation (RSD) was 0.70-4.09% and 0.60-4.81% for Cyt and 5mCyt, respectively. The intraday precision expressed as RSD ranged from 1.86% to 4.67%, whereas the interday values ranged from 3.72% to 4.68%. The recovery of the method varied from 86.52% to 105.14%. This yielded a simple and reliable LC-MS/MS assay for detection of Cyt and 5mCyt, thereby enabling the evaluation of global DNA methylation.

Simultaneous Determination of Global DNA Methylation and Hydroxymethylation Levels by Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry

Methylation of DNA at the 5-position of cytosine (Cyt) is a well-studied epigenetic pathway implicated in gene silencing and embryogenesis. Recently, in addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) have been detected in certain mammalian tissues. Here, we developed and validated a hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous determination of Cyt, 5mC, and 5hmC levels in biological samples. DNA was extracted with phenol-chloroform, hydrolyzed using 88% formic acid at 140 °C, separated using a bridged ethylene hybrid HILIC column, and analyzed by tandem MS. The linearity was established over the concentration range of 1 to 500 ng/mL for Cyt, 0.2 to 100 ng/mL for 5mC, and 0.1 to 50 ng/mL for 5hmC, and the correlation coefficients were all >0.99. Limits of detection were 1 pg/mL for Cyt, 45 pg/mL for 5mC, and 57 pg/mL for 5hmC, and the limit of quantification values for Cyt, 5mC, and 5hmC were 2 pg/mL, 90 pg/mL, and 100 pg/mL, respectively. The relative standard deviation (RSD) of the intraday precision ranged from 1.87% to 4.84% and the interday precision from 2.69% to 4.98%. The recovery of the method varied from 88.25% to 104.39%. The method was then applied to the analysis of DNA from biological samples, establishing its potential for helping researchers understand the roles of modified nucleobases in DNA.

Characteristics of fatty acid distribution is associated with colorectal cancer prognosis

To investigate tissue fatty acid distribution in relation to the incidence of colorectal cancer prognosis, adjacent normal tissue and cancerous tissue from 35 samples of clinically incident colorectal cancer were obtained. Fatty acids were measured in the colorectal mucosa phospholipid fraction by gas chromatography mass spectrometry. Palmitoleic acid and oleic acid were significantly lower in colorectal cancerous tissue, ranging from 20% to 50% less than the adjacent normal tissue. The omega-6 (n-6) fatty acid family members (20:2, 20:3, 20:4 and 22:4) were higher by 1-3 fold in cancerous colorectal tissue. Contrary with the high level of n-6 fatty acids, about a 37% to 87% reduction in EPA and DHA was observed in colorectal cancerous tissue. A higher level of linoleic acid and arachidonic acid was detected in the C cancer stage than in the B cancer stage (p<0.05), but a lower level of oleic acid and docosahexenoic acid was detected in the C cancer stage (p<0.05). The fatty acid distribution of colorectal tissue is strongly linked to the incidence of colorectal cancer. This study also provides scientific basis for identifying novel biomarkers for the diagnosis and treatment of cancer.

Quantification of the sixth DNA base 5-hydroxymethylcytosine in colorectal cancer tissue and C-26 cell line

BACKGROUND DNA methylation at the five position of cytosine is well recognized as an important epigenetic modification in human health and disease. Recent evidences demonstrated that 5-methylcytosine (5-mC) by the TET family of enzymes can be converted to 5-hydroxymethylcytosine (5-hmC). Here, we use an ultrasensitive and accurate isotope-based LC-MS/MS method to precisely determine the levels of 5-hmC and 5-mC in colorectal cancer and the C-26 colon adenocarcinoma cell line. RESULTS Our data showed that 5-hmC content is significantly reduced (approximately sixfold) in colorectal cancer as compared with adjacent normal tissue. Similarly, the ratio of 5-hmC to 5-mC dropped from 0.054 ± 0.005 in normal tissues, to 0.011 ± 0.002 in cancer. CONCLUSION The analysis of 5-hmC levels and the ratio of 5-hmC:5-mC during tumor progression might provide insight into the role of this modification in cellular immortalization and transformation.

Omega-3 Polyunsaturated Fatty Acids Inhibited Tumor Growth via Preventing the Decrease of Genomic DNA Methylation in Colorectal Cancer Rats.

ABSTRACT Omge-3 polyunsaturated fatty acids (PUFAs) exhibited significant effect in inhibiting various tumors. However, the mechanisms of its anticancer role have not been fully demonstrated. The declination of 5-methylcytosine (5 mC) was closely associated with poor prognosis of tumors. To explore whether omega-3 PUFAs influences on DNA methylation level in tumors, colorectal cancer (CRC) rat model were constructed using N-methyl phosphite nitrourea and omega-3 PUFAs were fed to part of the rats during tumor induction. The PUFAs contents in the rats of 3 experimental groups were measured using gas chromatography and 5 mC level were detected by liquid chromatography tandem mass spectrometry. The results showed that tumor incidence in omega-3 treated rats was much lower than in CRC model rats, which confirmed significant antitumor role of omega-3 PUFAs. Six PUFA members categorized to omega-3 and omega-6 families were quantified and the ratio of omega-6/omega-3 PUFAs was remarkably lower in omega-3 PUFAs treatment group than in CRC model group. 5 mC content in omega-3 PUFAs treated rats was higher than in CRC model rats, suggesting omega-3 PUFAs promoted 5 mC synthesis. Therefore, omega-3 PUFAs probably inhibited tumor growth via regulating DNA methylation process, which provided a novel anticancer mechanism of omega-3 PUFAs from epigenetic view.

A quantitative method for detecting DNA methylation over targeted genomic regions using isotope dilution liquid chromatography tandem mass spectrometry

Aberrant DNA methylation is associated with various diseases. Quantitative analysis of regional DNA methylation levels of some specific genes would aid in diseases diagnosis and risk stratification. In this study, we developed a robust method for detecting DNA methylation level over targeted genomic regions using nucleobases quantification in bisulfite amplicons by isotope dilution liquid chromatography tandem mass spectrometry coupled with a simple equation. This method had wide detection range (from 0% to 100% methylation) and high accuracy while more time-saving compared to clonal bisulfite sequencing method. The application for clinical tissue samples showed good applicability and cost effectiveness. This analytical method is suitable for quantifying average DNA methylation level over targeted genomic regions and expected to be a useful tool for detecting DNA methylation biomarkers.

Validation and quantification of genomic 5-carboxylcytosine (5caC) in mouse brain tissue by liquid chromatography-tandem mass spectrometry

5-Carboxylcytosine (5caC) is one of the most important oxidation products of 5-methylcytosine, an epigenetic biomarker generated from cytosine (Cyt). Although several methods have been developed to detect 5caC, they still can not accurately quantify trace amounts of 5caC. To conquer this challenge, we developed and validated a simple, robust method for the quantification of 5caC levels in mammalian tissue by LC-MS/MS. Tissue DNA was isolated using a commercial kit, hydrolyzed using 88% formic acid at 140 °C, separated using a bridged ethylene hybrid HILIC column, and analyzed by tandem MS. The linearity was evaluated in the concentration range of 40 to 4000 ng mL−1 for Cyt and 1 to 100 ng mL−1 for 5caC, and both the correlation coefficients were higher than 0.99. The limits of detection were 0.05 ng mL−1 for Cyt and 0.1 ng mL−1 for 5caC, and the limits of quantification were 0.1 ng mL−1 for Cyt and 1 ng mL−1 for 5caC. All the relative standard deviation (RSD) values of intra-day precision were lower than 6%. The recovery of the method ranged from 93.42% to 96.54% with RSD lower than 0.6%. Using this method, we illustrated that 5caC was distributed in mouse brain tissue, and the content of 5caC in the cerebrum was higher than that in the cerebellum and brainstem. Our studies indicated that the LC-MS/MS method was adequate for analyzing 5caC levels in biological samples.

Application of LC-MS/MS to the searching of methylated exons in colorectal cancer tissues

Abstract Tumor suppressor genes (TSGs) with DNA methylation has been suggested as effective biomarkers for cancer identification and promoter methylation in TSGs has been widely discussed. However, the feasibility of TSG exon methylation used for discriminating cancers was not fully clarified yet. In this study, genomic DNA was isolated from colorectal tissues and responding cancer-adjacent counterparts, and then bisulfite-converted. The exon 1 region of four popular TSGs including ALX4, FBN1, MLH1 and BCL2 was amplified from bisulfite-treated DNA, using nest- polymerase chain reaction (PCR) technique. The purified amplicon was hydrolyzed and then used to detect the methylation level using a robust and convenient LC-MS/MS method. The methodological validation revealed the favorable sensitivity and accuracy of established LC-MS/MS approach. The LC-MS/MS result showed that the methylation level of ALX4 and FBN1 exon in colorectal cancerous tissues (23.70 ± 10.85 and 33.23 ± 6.64) was significant higher than that in cancer-adjacent tissues (12.15 ± 7.08 and 22.08 ± 4.46) with statistic difference, and their value of area under curve (AUC) in receiver operator characteristic curve (ROC) analysis were higher than 0.8. On the other hand, the methylation level of MLH1 and BCL2 was 13.95 ± 6.93 and 15.46 ± 2.41 in CRC tissues and 13.87 ± 3.47 and 12.84 ± 1.91 in cancer-adjacent tissues, respectively. It is demonstrated that the methylation alteration of MLH1 and BCL2 exhibited a non-differential association between two groups. The finding indicated exon methylation of ALX4 and FBN1 could effectively distinguish CRC from non-CRC tissue and exon-based methylation should be used as potential biomarkers for cancer identification. Graphical Abstract

The hypermethylation of p16 gene exon 1 and exon 2: potential biomarkers for colorectal cancer and are associated with cancer pathological staging

BackgroundTumor suppressor gene p16 promoter hypermethylation has been widely studied in colorectal cancer (CRC), yet its clinicopathological significance remains controversial. The methylation alterations of other regions within p16 gene are still rarely researched. The present study aimed to explore the methylation changes of p16 gene body in CRC and to find whether they were associated with clinicopathological staging of CRC.MethodsPaired colorectal cancer tissues and corresponding adjacent normal tissues from 30 CRC patients were collected. The methylation levels of two CpG islands within p16 gene body, exon 1 and exon 2, were accurately assessed simultaneously by a LC-MS/MS method. The p16 protein expressions were assessed by immunohistochemistry assay. Statistical analyses were carried out using SPSS 17.0 software. Heat-map analysis was carried out by HemI 1.0 software.ResultsIn the present study, CRC tissues showed more highly methylated than adjacent normal tissues at both CpG islands of p16 gene. And exon 2 hypermethylation was higher and more frequent than exon 1. The ROC curve analysis showed that the simultaneous use of both indicators had excellent sensitivity and specificity for distinguishing CRC tissues and adjacent normal tissues. Following, the methylation level of p16 exon 1/2 was negatively related to p16 protein expression. Further correlation analysis revealed that p16 exon 1 hypermethylation was associated with N/Dukes staging (p = 0.033), and p16 exon 2 hypermethylaiton was associated with T staging (p = 0.035).ConclusionsThe p16 gene body was remarkably hyper-methylated in CRC tissues and associated with p16 protein expression and cancer clinicopathological staging. The combination of p16 exon 1 and exon 2 could better reflect the overall methylation status of p16 gene body and provide potential biomarkers of CRC.

The anticancer role of omega-3 polyunsaturated fatty acids was closely associated with the increase of genomic DNA hydroxymethylation

BACKGROUND Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) have significant multiple anti-tumor roles. However, whether epigenetic DNA hydroxymethylation enroll in the anticancer process of omega-3 PUFAs is still not clear yet. OBJECTIVE To expound the interaction between anti-tumor role of omega-3 PUFAs and the DNA demethylation pathway and thus provide a firm foundation for deepening our understanding on anticancer mechanism of omega-3 PUFAs. METHODS Colorectal cancer (CRC) model rats were induced to generate tumor by N-methyl-N-nitrosourea and their counterparts treated with omega-3 PUFAs during the induction. The blood samples from different treatment groups of rats [normal control group (NC), colorectal cancer model group (CRC) and omega-3 PUFAs medication group (MG)] were used as experimental materials. Genomic 5-hydroxymethylocytosine (5hmC) content was quantified using LC-MS/MS, and the expression of ten-eleven translocation dioxygenase 1 (TET1), who catalyzed the generation of 5hmC, was also evaluated by quantitative real-time PCR and western-blotting. RESULTS We observed lower tumor incidence and small tumor size in MG group when compared with CRC group, supporting the effective anticancer role of omega-3 PUFAs. Due to the formation of CRC, 5hmC level was dramatically dropped in CRC group when compared with NC group. Notably, 5hmC percentage in MG group remarkably increased to close to NC group and was significantly higher than that in CRC group. Consistent alteration pattern of TET1 expressions in mRNA and protein levels was also observed in the tested groups of rats. CONCLUSION The anticancer effect of omega-3 PUFAs was positively correlated with global 5hmC accumulation and TET1 expression, suggesting DNA hydroxymethylation pathway was factually involved in the anticancer process of omega-3 PUFAs.