The Biochemical Impact by Covalent Shielding of the Anionic Oxygen of the Phosphate Group in DNA and RNA as Methylated Phosphotriester Linkage on the Inhibition of DNA Duplication and on HIV-1 RNA Viral Infectivity Has Been Seriously Overlooked

With the help of model experiments, we are able to offer a detailed proposal for the inhibition of DNA duplication and no inhibition of RNA viral infectivity. As a backbone, we introduced methyl phosphotriester (MPTE). Duplex formation according to the traditional Watson and Crick base-pairing: [(MPTE)n−1 DNA] * DNA and [(MPTE)n−1 DNA] * RNA, where n = number of DNA and RNA bases. However, in the latter case, inhibition is obtained by reduction of the number of MPTE linkages, as is confirmed with model experiments and under biological conditions with micro (mi)RNA substrates. The latter results have recently been published. One or more single MPTEs are disseminated over different places of DNA without neighbour MPTEs (Prof. Wen-Yih Chen and his group, Taiwan).

Diagnostic value of methylated branched chain amino acid transaminase 1/IKAROS family zinc finger 1 for colorectal cancer

BACKGROUND The diagnostic value of combined methylated branched chain amino acid transaminase 1(BCAT1)/IKAROS family zinc finger 1(IKZF1)in plasma for colorectal cancer(CRC)has been explored since 2015.Recently,several related studies have published their results and showed its diagnostic efficacy.AIM To analyze the diagnostic value of methylated BCAT1/IKZF1 in plasma for screening and postoperative follow-up of CRC.METHODS The candidate studies were identified by searching the PubMed,Embase,Cochrane Library,CNKI,and Wanfang databases from May 31,2003 to June 1,2023.Sensitivity,specificity,and diagnostic accuracy were calculated by merging ratios or means.RESULTS Twelve eligible studies were included in the analysis,involving 6561 participants.The sensitivity of methylated BCAT1/IKZF1 in plasma for CRC diagnosis was 60%[95%confidence interval(CI)53-67]and specificity was 92%(95%CI:90-94).The positive and negative likelihood ratios were 8.0(95%CI:5.8-11.0)and 0.43(95%CI:0.36-0.52),respectively.Diagnostic odds ratio was 19(95%CI:11-30)and area under the curve was 0.88(95%CI:0.85-0.91).The sensitivity and specificity for CRC screening were 64%(95%CI:59-69)and 92%(95%CI:91-93),respectively.The sensitivity and specificity for recurrence detection during follow-up were 54%CONCLUSION The detection of methylated BCAT1/IKZF1 in plasma,as a non-invasive detection method of circulating tumor DNA,has potential CRC diagnosis,but the clinical application prospect needs to be further explored.

Clinical value of preoperative methylated septin 9 in Chinese colorectal cancer patients

BACKGROUND The methylated septin 9(mSEPT9) assay was the first blood-based test approved by the United States Food and Drug Administration as a colorectal screening test.However, the diagnostic and prognostic role of preoperative mSEPT9 for colorectal cancer(CRC) in Chinese patients is still unknown.AIM To improve the understanding of diagnostic and prognostic factors, serum m SEPT9 was detected in Chinese CRC patients.METHODS A retrospective analysis of 354 cases, of which 300 had CRC and 54 were normal,was performed in China. Patients' characteristics, treatments, and laboratory data, including age, the date of surgery, Union for International Cancer Control(UICC) stages, distant metastasis(M), and so on, were collected. Methylation levels of SEPT9 were quantified by quantitative, methylation-specific polymerase chain reaction before surgery. In addition, the effects of mSEPT9 on the occurrence and prognosis of 330 CRC cases from The Cancer Genome Atlas(TCGA) database were evaluated using bioinformatics analyses. Potential prognostic factors for overall survival(OS) and progression-free survival(PFS)were evaluated by Kaplan-Meier univariate analysis.RESULTSIn Chinese CRC patients, positive mSEPT9 was strongly associated with advanced UICC stages, deeper invasion by the primary tumor, and more distant metastasis. Methylation levels of SEPT9 were stage-dependent and showed a stepwise increase in UICC stages(I–IV), primary tumor categories(T1–T4),regional node categories(N0–N2), and distant metastasis categories(M0–M1).The patients with positive mSEPT9 showed a tendency toward lower PFS. After analyzing TCGA clinical data, the high mSEPT9 group was found to be obviously correlated only with more distant metastasis. The patients with high mSEPT9 levels showed a tendency toward lower OS. Besides, nine meaningful mSEPT9 sites were found to provide guidance for the follow-up studies.CONCLUSION MSEPT9 analysis may add valuable information to current tumor staging. Serum m SEPT9 in Chinese CRC patients appears to offer promising novel prognostic markers and might be considered for monitoring CRC recurrence.

A differentially methylated region of the DAZ1 gene in spermatic and somatic cells

Aim： To investigate the methylation status of the deleted in azoospermia 1（DAZ1） gene promoter region in different cell types. Methods： Using CpG island Searcher software, a CpG island was found in the promoter region of the DAZ1 gene. The methylation status of this region was analyzed in sperm and leukocytes by bisulfited sequencing. Results： The methylation status of the CpG island in the DAZ1 gene promoter region differed in leukocytes and sperm： it was methylated in leukocytes, but unmethylated in sperm. Conclusion： A differentially methylated region of the DAZ1 gene exists in spermatic and somatic cells, suggesting that methylation of this region may regulate DAZ1 gene expression in different tissues. （Asian J Androl 2006 Jan; 8：61-67 ）

Synthesis of Methylated β-Cyclodextrins Derived Optically Active Poly(N-diphenylmethyl maleimide)

The methylated β-cyclodextrins derived optically active polymers are firs synthesized through asymmetric polymerization of N-diphenylmethyl maleimide with lithium salt of heptakis(2,6-O-dimethyl) β-cyclodextrin (DM-β-CD) as an initiator. The resulting polymers show negative specific rotation which is opposite in sign to that of DM-β-CD. The asymmetric induction is further confirmed by circular dichroism. The structure is characterized by IR and NMR spectroscopies

Effects of Methylated Soybean Oil Adjuvant on Fomesafen Efficacy to Weeds

Tank-mix adjuvant has the potential to improve the weed control efficacy of post-emergence herbicides. In order to study the synergistic effect of adjuvant, the effects of different rates of fomesafen alone or applied methylated soybean oil adjuvant（MSO） were sprayed on redroot pigweed, abutilon and black nightshade under greenhouse condition. The results showed that fomesafen had different performance on the three weeds, and MSO adjuvant could effectively increase the control. The nightshade control was lower than other two weeds with all the fomesafen doses from 131.25 to 506.25 ga.i. · hm-2 with or without adjuvant. The control of abutilon was between the black nightshade and the redroot pigweed, and had better control at 375 ga.i. · hm-2 with adjuvant or 506.25 ga.i. · hm-2 alone or with adjuvant respectively. The results indicated that mixing adjuvant with fomesafen improved the control on weeds, especially at the low rate. Black nightshade was more difficult to control. The redroot pigweed had the most susceptibility to fomesafen alone or with adjuvant.

Hypermethylated SFRP2 gene in fecal DNA is a high potential biomarker for colorectal cancer noninvasive screening

AIM: To investigate the feasibility of detecting hypermethylated secreted frizzled-related protein 2 (SFRP2) gene in fecal DNA as a non-invasive screening tool for colorectal cancer (CRC). METHODS: Fluorescence-based real-time PCR assay (MethyLight) was performed to analyze SFRP2 gene promoter methylation status in a blinded fashion in tumor tissues and in stool samples taken from 69 CRC patients preoperatively and at the 9th postoperative day,34 patients with adenoma ≥ 1 cm,26 with hyperplastic polyp,and 30 endoscopically normal subjects. Simultaneously the relationship between hypermethylation of SFRP2 gene and clinicopathological features was analyzed. RESULTS: SFRP2 gene was hypermethylated in 91.3% (63/69) CRC,79.4% (27/34) and 53.8% (14/26) adenoma and hyperplastic polyp tissues,and in 87.0% (60/69),61.8% (21/34) and 42.3% (11/26) of corresponding fecal samples,respectively. In contrast,no methylated SFRP2 gene was detected in mucosal tissues of normal controls,while two cases of matched fecal samples from normal controls were detected with hypermethylated SFRP2. A significant decrease (P < 0.001) in the rate of hypermethylated SFRP2 gene was detected in the postoperative (8.7%,6/69) fecal samples as compared with the preoperative fecal samples (87%,60/69) of CRC patients. Moreover,no significant associations were observed between SFRP2 hypermethylation and clinicopathological features including sex,age,tumor stage,site,lymph node status and histological grade,etc. CONCLUSION: Hypermethylation of SFRP2 gene in fecal DNA is a novel molecular biomarker of CRC and carries a high potential for the remote detection of CRC and premalignant lesions as noninvasive screening method.

Silencing SMYD3 in hepatoma demethylates RIZI promoter induces apoptosis and inhibits cell proliferation and migration

AIM： To investigate the role of SMYD3 in hepatocellular carcinoma （HCC） development and progression and to verify whether its regulation activity was through RIZ1 inactivation. METHODS： Expression of SMYD3 in HCC cell lines and tissues were measured; silencing of SMYD3 by RNA interference （RNAi） was effectuated, hepatoma cell proliferation, migration and apoptosis were tested, with RIZl CpG promoter methylation, and corresponding mRNA expression were investigated. RESULTS： SMYD3 over-expression in HCC was associated with RIZl hypermethylation and mRNA down-expression. Suppression of SMYD3 expression de- methylated RIZl CpG promoter （P 〈 0.01） and increased RIZl mRNA expression （P 〈 0.01）. Consequently, SMYD3 down-expression with RIZl de-methylation strongly inhibited hepatoma cell growth （MTT inhibitory rates： Pgenesil-1-s1 60.95%± 7.97%, Pgenesil-1-s2 72.14% ± 9.68% vs Pgenesil-1-hk 6.89% ± 4.12%, P 〈 0.01） and migration （Pgenesil-1-s1 4.24% ± 1.58%, Pgenesil- 1-s1 4.87% ± 0.73% vs Pgenesil-1 19.03% ± 4.63%, Pgenesil-1-hk 19.95% ±5.21%, P 〈 0.01） and induced apoptosis （FCM subG1 phase Pgenesil-1-s1 19.07% ＋ 1.78%, Pgenesil-1-s2 17.68% ± 2.36% vs Pgenesil-1 0.47% ± 0.12%, Pgenesil-1-hk 1.46% ± 0.28%, P 〈 0.01. TUNEL-positive cells： Pgenesil-1-s1 40.24%± 5.18%, Pgenesil-1-s2 38.48% ± 4.65% vs Pgenesil-1 2.1B% - 1.34%, Pgenesil-1-hk 2.84%± 1.22%, P 〈 0.01） in HepG2 cells. CONCLUSION： These results demonstrate that SMYD3plays a critical role in the carcinogenesis and progression of HCC, The proliferation, migration induction and apoptosis inhibition activities of SMYD3 may be mediated through RIZl CpG promoter hypermethylation.

Bioinformatics analysis of aberrantly methylated-differentially expressed genes and pathways in hepatocellular carcinoma

AIM To discover methylated-differentially expressed genes(MDEGs) in hepatocellular carcinoma(HCC) and to explore relevant hub genes and potential pathways. METHODS The data of expression profiling GSE25097 and methylation profiling GSE57956 were gained from GEO Datasets. We analyzed the differentially methylated genes and differentially expressed genes online using GEO2 R. Functional and enrichment analyses of MDEGs were conducted using the DAVID database. A protein-protein interaction(PPI) network was performed by STRING and then visualized in Cytoscape. Hub genes were ranked by cytoH ubba, and a module analysis of the PPI network was conducted by MCODE in Cytoscape software. RESULTS In total, we categorized 266 genes as hypermethylated, lowly expressed genes(Hyper-LGs) referring to endogenous and hormone stimulus, cell surface receptor linked signal transduction and behavior. In addition, 161 genes were labelled as hypomethylated, highly expressed genes(Hypo-HGs) referring to DNA replication and metabolic process, cell cycle and division. Pathway analysis illustrated that Hyper-LGs were enriched in cancer, Wnt, and chemokine signalling pathways, while Hypo-HGs were related to cell cycle and steroid hormone biosynthesis pathways. Based on PPI networks, PTGS2, PIK3 CD, CXCL1, ESR1, and MMP2 were identified as hub genes for Hyper-LGs, and CDC45, DTL, AURKB, CDKN3, MCM2, and MCM10 were hub genes for Hypo-HGs by combining six ranked methods of cytoH ubba. CONCLUSION In the study, we disclose numerous novel genetic and epigenetic regulations and offer a vital molecular groundwork to understand the pathogenesis of HCC. Hub genes, including PTGS2, PIK3 CD, CXCL1, ESR1, MMP2, CDC45, DTL, AURKB, CDKN3, MCM2, and MCM10, can be used as biomarkers based on aberrant methylation for the accurate diagnosis and treatment of HCC.

Cell-free plasma hypermethylated CASZ1, CDH13 and ING2 are promising biomarkers of esophageal cancer

Identifying sensitive and specific biomarkers for early detection of cancer is immensely imperative for early diagnosis and treatment and better clinical outcome of cancer patients. This study aimed to construct a specific DNA methylation pattern of cancer suppressor genes and explore the feasibility of applying cell-free DNA based methylation as a biomarker for early diagnosis of esophageal squamous cell carcinoma(ESCC). We recruited early stage ESCC patients from Yangzhong County, China. The Illumina Infinium 450 K Methylation BeadChip was used to construct a genome-wide DNA methylation profile. Then, differentiated genes were selected for the validation study using the Sequenom MassARRAY platform. The frequency of methylation was compared between cancer tissues, matched cell-free DNAs and normal controls. The specific methylation profiles were constructed, and the sensitivity and specificity were calculated. Seven CG sites in three genes CASZ1, CDH13 and ING2 were significantly hypermethylated in ESCC as compared with normal controls. A significant correlation was found between the methylation of DNA extracted from cancer tissues and matched plasma cell-free DNA, either for individual CG site or for cumulative methylation analysis. The sensitivity and specificity reached 100% at an appropriate cut-point using these specific methylation biomarkers. This study revealed that aberrant DNA methylation is a promising biomarker for molecular diagnosis of esophageal cancer. Hypermethylation of CASZ1,CDH13 and ING2 detected in plasma cell-free DNA can be applied as a potential noninvasive biomarker for diagnosis of esophageal cancer.

XAF1 is frequently methylated in human esophageal cancer

AIM： To explore epigenetic changes in the gene encod- ing X chromosome-linked inhibitor of apoptosis-associ- ated factor 1 （XAF1） during esophageal carcinogenesis. METHODS： Methylation status of XAF1 was detected by methylation-specific polymerase chain reaction （MSP） in four esophageal cancer cell lines （KYSE30, KYSE70, BICl and partially methylated in TE3 cell lines）, nine cases of normal mucosa, 72 cases of pri- mary esophageal cancer and matched adjacent tissue. XAF1 expression was examined by semi-quantitative reverse transcriptional polymerase chain reaction and Western blotting before and after treatment with 5-aza- deoxycytidine （5-aza-dc）, a demethylating agent. To investigate the correlation of XAF1 expression and methylation status in primary esophageal cancer, immu- nohistochemistry for XAF1 expression was performed in 32 cases of esophageal cancer and matched adjacent tissue. The association of methylation status and clini-copathological data was analyzed by logistic regression. RESULTS： MSP results were as follows： loss of XAF1 expression was found in three of four esophageal cell lines with promoter region hypermethylation （com- pletely methylated in KYSE30, KYSE70 and BIC1 cell lines and partially in TE3 cells）; all nine cases of normal esophageal mucosa were unmethylated; and 54/72 （75.00%） samples from patients with esophageal can- cer were methylated, and 25/72 （34.70%） matched adjacent tissues were methylated （75.00% vs 34,70%, z2 = 23.5840, P = 0.000）. mRNA level of XAF1 mea- sured with semi-quantitative reverse transcription poly- merase chain reaction was detectable only in TE3 cells, and no expression was detected in KYSE30, KYSE70 or BIC1 cells. Protein expression was not observed in KYSE30 cells by Western blotting before treatment with 5-aza-dc. After treatment, mRNA level of XAF1 was detectable in KYSE30, KYSE70 and BIC1 cells. Protein expression was detected in KYSE30 after treatment with 5-aza-dc. Immunohistochemistry was performed on 32 cases of esophageal cancer and adjacent tissue, and demonstrated XAF1 in the nucleus and cytoplasm. XAF1 staining was found in 20/32 samples of adjacent normal tissue but was present in only 8/32 samples of esophageal cancer tissue （Z2= 9.143, P = 0.002）. XAF1 expression was decreased in cancer samples compared with adjacent tissues. In 32 cases of esophageal can- cer, 24/32 samples were methylated, and 8/32 esopha- geal cancer tissues were unmethylated. XAF1 staining was found in 6/8 samples of unmethylated esophageal cancer and 2/24 samples of methylated esophageal cancer tissue. XAF1 staining was inversely correlated with XAF1 promoter region methylation （Fisher＇s exact test, P = 0.004）. Regarding methylation status and clinicopathological data, no significant differences were found in sex, age, tumor size, tumor stage, or metas- tasis with respect to methylation of XAF1 for the 72 tis- sue samples from patients with esophageal cancer. CONCLUSION： XAF1 is frequently methylated in eso- phageal cancer, and XAF1 expression is regulated by promoter region hypermethylation.

hsa-mir-183 is frequently methylated and related to poor survival in human hepatocellular carcinoma

To screen clinically relevant microRNAs (miRNAs) silenced by DNA methylation in human hepatocellular carcinoma (HCC).METHODSKnockdown of DNA methyltransferases (DNMTs) using siRNAs and miRNA profiling in HCC cell lines were performed to identify DNA hypermethylation-mediated miRNA downregulation. Confirmation using individual quantitative real-time PCR (qRT-PCR) assays was then performed followed by DNA methylation quantification at the promoter of the miRNA genes. Quantification of DNA methylation and miRNA expression was then performed in primary HCC tumor samples and related with clinicopathological variables.RESULTSmiRNA profiling after DNMT knockdown in HCC cell lines revealed upregulation of miR-23, miR-25 and miR-183. After qRT-PCR confirmation and CpG island methylation quantification of these miRNAs in cell lines, further analysis in primary HCC specimens showed that hsa-miR-183 is hypermethylated in 30% of HCC (n = 40). Expression of mature miR-183 showed an inverse correlation with DNA methylation levels. In HCC cells, DNMT knockdown and 5-aza-2’-deoxycytidine treatment reduced methylation and stimulated expression of miR-183. In HCC patients, hypermethylation at hsa-miR-183 promoter significantly correlates with poor survival (log-rank test P = 0.03). DNA methylation analysis in healthy liver, benign liver tumors (hepatocellular adenoma and focal nodular hyperplasia) and their corresponding adjacent tissues showed absence of hypermethylation supporting the notion that aberrant methylation at hsa-miR-183 is specific for the malignant transformation of hepatocytes.CONCLUSIONOur data indicate that hypermethylation of hsa-miR-183 is a frequent event in HCC and potentially useful as a novel surrogate diagnostic and prognostic marker.

Procaine Inhibiting Human Bladder Cancer Cell Proliferation by Inducing Apoptosis and Demethylating APAF1 CpG Island Hypermethylated

Studies have shown that aberrant DNA methylation of apoptotic protease activating factor-1（APAF1） is an important epigenetic mechanism of gene regulation in the progression of bladder cancer.In this article,we have proved that procaine,an inhibitor of DNA methyltransferases,could inhibit the proliferation of T24 and 5637 human bladder cancer cells by inducing their apoptosis.The mechanism studies reveal that procaine could induce demethylation of APAF1 gene in T24 or 5637 cells,subsequently activating caspase-3/9.It was also shown that the serum soluble fas ligand（sFasL） was activated,and the expression of matrix metallopeptidase 9（MMP-9） was down-regulated.Procaine seems to induce cell death by different pathways,and it might be used as a potential agent for bladder cancer treatment.

Combination analysis of hypermethylated SFRP1 and SFRP2 gene in fecal as a novel epigenetic biomarker panel for colorectal cancer screening

Objective：To investigate the feasibility of the combination of detecting hypermethylated secreted frizzled-related protein 1 （SFRP1） and secreted frizzled-related protein 2（SFRP2） in feces as a panel of biomarkers for colorectal cancer（CRC） screening. Methods： Methylation-specific PCR（MSP） was performed to analyze methylation status of SFRP1 and SFRP2 in a blinded fashion in tumor tissues and in matched stool samples from 39 patients with primary CRC, 34 patients with adenomas, 17 patients with hyperplastic polyps and 20 endoscopically normal subjects as normal controls. Simultaneously we analyzed the correlation of hypermethylated SFRP1 and SFRP2 with the clinicopathological features of CRC. Results：Hypermethylated SFRP1 was detected in 92.3%, 76.5%, 47.1% of tissue samples and in 89.7%, 64.7%, 35.3% of matched fecal samples from CRC, adenoma and hyperplastic polyp, respectively. Hypermethylated SFRP2 was detected in 87.2%, 67.6%, 35.3% of tissue samples and in 82.1%, 55.9%, 29.4% of matched fecal samples from CRC, adenoma and hyperplastic polyp, respectively. Of these two genes, at least one hypermethylated was 94.9%, 82.4%, 52.9% in tissue samples and 92.3%,73.5%, 47.1% in matched fecal samples from CRC, adenoma and hyperplastic polyp, respectively. In contrast, no hypermethylated SFRP1 and SFRP2 were detected in mucosa tissues of normal controls, only 2 cases of fecal samples was detected with hypermethylated SFRP1 and another 1 case was detected with hypermethylated SFRP2. Moreover, no significant associations were observed between hypermethylated SFRP1,SFRP2 and clinicopathological features of CRC. Conclusion： Hypermethylation of SFRP1 and SFRP2 in feces are novel epigenetic biomarkers of CRC and carded high potential for the remote detection of CRC as non-invasive screening method, and combined analysis of hypermethylated SFRP1 and SFRP2 in fecal could further increase the detection rate of CRC and premalignant lesions.

Kaiso mainly locates in the nucleus in vivo and binds to methylated,but not hydroxymethylated DNA

Objective： Kaiso is upregulated in many cancers and proposed to bind with both methylated- and unmethylated-DNA in the nucleus as a transcriptional repressor. The objective is to define its subcellnlar localization in vivo and exact binding DNA sequences in cells. Methods： Compartmentalization of exogenous Kaiso in cells was tracked with enhanced green fluorescence protein （EGFP） tag. The endogenous Kaiso expression in gastric carcinoma tissue was examined with immunohistochemical staining. Kaiso-DNA binding was tested using electrophoretic mobility shift assay （EMSA） and chromatin immunoprecipitation assay （CHIP）. Results： Kaiso mainly localized in the nucleus of cancer and stromal cells in vivo, but remained in the cytoplasm of cultured cells. Most importantly, nuclear Kaiso can bind with the methylated-CGCG- containing sequence in the CDKN2A promoter, but not with the hydroxymethylated-CGCG sequence in HCT116 cells. Conclusions： Kaiso locates mainly in the nucleus in vivo where it binds with the methylated-CGCG sequences, but does not bind with the hydroxymethylated-CGCG sequences.

Isolation and bioinformatics analysis of differentially methylated genomic fragments in human gastric cancer

AIM:To isolate and analyze the DNA sequences which are methylated differentially between gastric cancer and normal gastric mucosa. METHODS: The differentially methylated DNA sequences between gastric cancer and normal gastric mucosa were isolated by methylation-sensitive representational difference analysis (MS-RDA). Similarities between the separated fragments and the human genomic DNA were analyzed with Basic Local Alignment Search Tool (BLAST). RESULTS: Three differentially methylated DNA sequences were obtained, two of which have been accepted by GenBank. The accession numbers are AY887106 and AY887107. AY887107 was highly similar to the 11th exon of LOC440683 (98%), 3' end of LOC440887 (99%), and promoter and exon regions of DRD5 (94%). AY887106 was consistent (98%) with a CpG island in ribosomal RNA isolated from colorectal cancer by Minoru Toyota in 1999. CONCLUSION: The methylation degree is different between gastric cancer and normal gastric mucosa. The differentially methylated DNA sequences can be isolated effectively by MS-RDA.

Special DNA Methylated Sites Between Haploid of Twin-Seedling and Its Hybrids in Rice(Oryza sativa)

DNA methylation is one of the epigenetic phenomena which can be transferred to the offspring by cell division in the evolution of organisms. The epigenetic regulation accompanied by gene expression can be found directly in the phenotype of haploidy plants. DNA cytosine methylation at the 5＇-CpCpGpG sites of haploid, Shuhui 527, Shuhui 363 and their hybrids was analyzed by methylation sensitive amplification polymorphism （MSAP） method. There were 765 DNA methylated sites detected and the methylation level was lower in hybrids than parents. Meanwhile, the different bands between hybrids and parents were analyzed and two types of methylated sites were detected, of which one inherited from haploid, and the other did not. The biological functions of genes related to methylated sites involved in cell structure, metabolize and response factor. Therefore, DNA methylated modifications can activate and silence the genes and play an important role in plant growth, development and evolution.

Droplet digital polymerase chain reaction assay for methylated ring finger protein 180 in gastric cancer

BACKGROUND Gastric cancer(GC)is one of the most prevalent malignant tumors that endangers human health.Early diagnosis is essential for improving the prognosis and survival rate of GC patients.Ring finger protein 180(RNF180)is involved in the regulation of cell differentiation,proliferation,apoptosis,and tumorigenesis,and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC.Thus,methylated RNF180 can be used as a potential biomarker for GC diagnosis.AIM To use droplet digital polymerase chain reaction(ddPCR)to quantify the methylation level of the RN180 gene.A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.METHODS The primer and probe were designed and selected,the conversion time of bisulfite was optimized,the ddPCR system was adjusted by primer concentration,amplification temperature and amplification cycles,and the detection limit of ddPCR was determined.RESULTS The best conversion time for blood DNA was 2 h 10 min,and that for plasma DNA was 2 h 10 min and 2 h 30 min.The results of ddPCR were better when the amplification temperature was 56°C and the number of amplification cycles was 50.Primer concentrations showed little effect on the assay outcome.Therefore,the primer concentration could be adjusted according to the reaction system and DNA input.The assay required at least 0.1 ng of input DNA.CONCLUSION In summary,a ddPCR assay was established to detect methylated RNF180,which is expected to be a new diagnostic biomarker for GC.

Comparison of next generation sequencing-based and methylated DNA immunoprecipitation-based approaches for fetal aneuploidy non-invasive prenatal testing

Over the past few years, many researchers have attempted to develop non-invasive prenatal testing methods in order to investigate the genetic status of the fetus. The aim is to avoid invasive procedures such as chorionic villus and amniotic fluid sampling, which result in a significant risk for pregnancy loss. The discovery of cell free fetal DNA circulating in the maternal blood has great potential for the development of non-invasive prenatal testing(NIPT) methodologies. Such strategies have been successfully applied for the determination of the fetal rhesus status and inherited monogenic disease but the field of fetal aneuploidy investigation seems to be more challenging. The main reason for this is that the maternal cell free DNA in the mother's plasma is far more abundant, and because it is identical to half of the corresponding fetal DNA. Approaches developed are mainly based on next generation sequencing(NGS) technologies and epigenetic genetic modifications, such as fetal-maternal DNA differential methylation. At present, genetic services for non-invasive fetal aneuploidy detection are offered using NGS-based approaches but, for reasons that are presented herein, they still serve as screening tests which are not readily accessed by the majority of couples. Here we discuss the limitations of both strategies for NIPT and the future potential of the methods developed.

Different denaturation rates between methylated and non-methylated genomic DNA can result in allele-specific PCR amplification

We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treated DNA to be on the methylated allele in the unaffected father and the unmethylated allele in the affected child. PCR from genomic DNA was then carried out using a commercial PCR kit with its recommended initial DNA denaturation step of 2 minutes. Subsequent sequence analysis showed that only the non-methylated allele had been amplified, the father appearing to be homozygous normal and the child appearing to have a homozygous 7 b.p. insertion. The PCR protocol was then modified in order to use a longer DNA denaturation stage prior to the addition of the polymerase enzyme. Upon doing so, both the methylated and non-methylated alleles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methylation status of DNA can affect the denaturation rate prior to PCR and result in allele drop-out, showing that the standard protocols of commercial kits should be used with caution when working with methylated regions of DNA.