| Part Ⅰ Analysis of DNA methylation characteristics of bladder cancer tissue in TCGA databaseObjectives:At present,many studies have shown that DNA methylation,as an epigenetic feature,is closely related to the occurrence and development of human tumors[1].In this part of the study,by comparing the DNA methylation data of bladder cancer tissue and normal bladder tissue provided in the Cancer Genome Atlas(TCGA)database,we explored tumor-specific methylation modification sites of bladder cancer,and established a predictive tool for plasma DNA methylation analysis of microcirculation tumor markers.Methods:We searched and downloaded the DNA methylation data of bladder cancer and normal bladder tissue required for this experiment from the TCGA database(https://cancergenome.nih.gov/).We chose the Illumina Human Methylation450 chip as the data set.We selected the DNA methylation data of 412 bladder cancer tissues and 21 normal tissues.We used the statistical method of analysis of variance and TukeyHSD multiple comparison to analyze these data and identified 14132 bladder cancer specific methylation modification sites(p<0.001).We obtained peripheral blood leukocytes full DNA methylation sequencing data from the Blueprint Epigenome database to eliminate white blood cell background noise in cfDNA and find out specific methylation markers that can be used for early diagnosis of bladder cancer.Results:We analyzed the DNA methylation sequencing data of 412 bladder cancer tissues and 21 normal tissues obtained from the TCGA database.The analysis results showed that there is a significant difference in DNA methylation levels in tissues and blood between patients with bladder cancer and normal people.At the same time,we also found that a total of 7752(53.44%)hypermethylation occurred in all specific methylation modification sites,of which 6325 occurred on CpG islands and 1227 occurred on flanks.Conclusions:The results of a macro-comparison of methylation sequencing data obtained from the TCGA database show that there is a significant difference in DNA methylation be-tween bladder cancer patients and normal people in their tissues.We identified some differentially methylated CpG site(DMC or DMS)between bladder cancer patients and normal human tissue samples.These sites may become markers for early screening of bladder cancer.Part Ⅱ Targeting capture sequencing analysis of cfDNA in bladder cancer patients’ peripheral blood by high-throughput sequencingObjectives:In the first phase of the study,we have found some CpG sites through the TCGA da-tabase.These sites may be potential markers for early screening of bladder cancer.In this part of the study,we established a high-throughput sequencing and analysis method for cfDNA methylation by analyz-ing the bladder cancer DNA methylation markers obtained from the sequencing in-formation in the TCGA database.We used this method to specifically analyze pe-ripheral blood DNA methylation in patients with bladder cancer in our center.At the same time,We set the peripheral blood DNA methylation of healthy people as a control group to analyze the difference in cfDNA methylation between two groups.Methods:We selected 10 cases of bladder cancer patients diagnosed by pathology in Nanfang Hospital from 2010 to 2017(including 3 cases of bladder cancer after kidney transplantation)and 10 healthy persons,and conducted targeted cfDNA methylation experiment analysis.There were no statistical differences between the 10 bladder cancer patients and 10 healthy people in terms of gender,age,height and weight.After obtaining the subject’s consent and signing the informed consent form,each subject draws ap-proximately 10 ml of venous blood.We centrifuged the blood after extraction,sepa-rated the serum,and extracted the cfDNA with magnetic beads.Based on the experimental results of the first stage,we established a specific cfDNA methylation library and completed high-throughput methylation sequencing with sequencing equipment.Results:We selected 10 patients with bladder cancer diagnosed by pathology and 10 healthy people as experimental subjects,and took venous blood samples for methylation high-throughput targeted sequencing capture.We obtained 14.6GB of sequencing data.Next,we analyzed and compared the cfDNA methylation data of bladder cancer patients and healthy people samples,and finally obtained 1295 DMC.The results showed a difference in methylation between bladder cancer samples and normal samples.As the p-value decreased,the difference in methylation between samples became more significant.Secondly,we conducted hierarchical clustering analysis and subtype association analysis of DMC methylation in patients with bladder cancer.Most DMC methylation levels can be clustered together according to their tumor stage in cluster analysis.At the same time,some methylation modification sites appear to be up-regulated during tumor development,and some methylation modification sites may appear to be down-regulated during tumor development.Conclusions:There are differential methylation CpG sites between plasma cfDNA of bladder cancer patients and healthy people.There are also differences in the degree of DMC methylation up and down regulation of plasma cfDNA of bladder cancer in different stages.These differentially methylated CpG sites may become new bladder cancer screening,staging and prognostic markers. |
