| Background and purpose:Gastric cancer is the fourth most malignant tumor in the world,and the second is the most fatal disease.The cases in East Asia account for more than half of the total.From the histological classification of gastric cancer,there are mainly adenocarcinoma,adenosquamous carcinoma,squamous cell carcinoma,carcinoid,etc.,most of which are gastric adenocarcinoma.Although gastric cancer can be found through physical examination,imaging and endoscopic biopsy,most patients with gastric cancer in China are found to have a late clinical stage at the time of detection,and the proportion of advanced gastric cancer is about 70%,which seriously afects the prognosis.Therefore,it is of great significance to study the related pathogenic mechanism of gastric malignant tumor.Epigenetics is a discipline in which the phenotype of DNA can be passed on to the next generation without changing the DNA base sequence.DNA methylation is one of its classic regulation and plays an important role in the regulation of various diseases.In recent years,5hmC,as an intermediate product of the cytosine modification process,has enriched our understanding of the process of epigenetic regulation of DNA methylation,and also provided us with new clues to search for genes related to gastric cancer.However,there are few studies on the whole genome 5hmC in gastric cancer.In this study,we will study the expression of 5hmC in gastric cancer through hMe-Seal chemical labeling combined with high-throughput sequencing,and explore the pathogenic mechanism of specific 5hmC distribution affecting the course of gastric cancer.Method1.Collect 6 cases of gastrointestinal surgery from the Second Hospital of Jilin University from January 2017 to January 2018.The surgically resected specimens of patients with pathological diagnosis of gastric cancer,each group of specimens includes tumor tissues,adjacent tissues,and normal distal tissues.2.Extract the DNA using phenol-chloroform method,and then quantify 5hmC by dot blot method.3.Use 5hmC-specific chemical imprinting(hME-seal)to label 5hmC for enrichment and purification.4.Perform library construction,high-throughput sequencing on the obtained 5hmC,perform quantitative and qualitative analysis,and look for differential hydroxymethyl(DhMRs).5.Position DhMRs to the human gene hg19 for bioinformatics analysis,use t-p-n as the stage of tumor progression to analyze tumor evolution,and explore the relationship between DhMRs and gastric cancer superenhancers.Result1.It was determined by dot blot that compared with normal gastric tissues,5hmC showed a significant decrease(P<0.001).The average content of 5hmC in gastric cancer was only about 35%of normal tissues.In normal gastric tissues and adjacent tissues In comparison,there was no significant change between the two(P>0.001).2.The high-throughput sequencing method(hME-seal-seq method)after enrichment using chemical labeling method showed that the expression level of 5hmC showed a downward trend in tumor tissues compared with normal tissues;in tissues adjacent to cancer and normal tissues,5hmC expression level has an upward trend.From the above results,we believe that the expression level of 5hmC decreases in gastric cancer,and the decrease in the expression level of 5hmC can become a specific marker of gastric cancer.3.By normalizing the reading count,estimating the dispersion and performing the difference test on the diferential hydroxymethylated CPG sites,the sites with P<0.01 are determined as the DhMRs area.The results showed that 17,318 gene loci showed a decrease of 5hmC,and 1344 gene loci showed a rise of 5hmC.In the tissues adjacent to cancer and normal tissues,a total of 4795 gene loci increased and 1876 gene loci decreased.And found that the DhMRs region is mainly distributed within the gene in the genome,but decreased in the intergenic region.4.Map DhMRs to hg 19 human reference sequence to find the corresponding genes,and further analyze the GO pathway of these genes to find that DhMRs are involved in multiple tumor pathways,and there are obvious differences between tumor tissues and adjacent tissues.We speculate that the unique changes of 5hmC DhMRs related to the surrounding tissues and tumor tissues may reflect diferent stages of tumorigenesis.We used phantom analysis to analyze the binding sites of potential transcription factors for the detected DhMRs.The results showed that the hypoxia-inducible factor 1(HIF1)was combined in the common motif of DhMRs in tumor tissues and adjacent tissues.In addition,Myc binding sites increased in tumor tissue DhMRs.These results suggest that the change of 5hmC in the occurrence and development of gastric cancer may lead to changes in the binding of HIF1 and Myc to DNA.5.Using t-p-n as the tumor progression stage,we performed a tumor evolution analysis.Our results showed a diferent set of DhMRs.There were 74 gene loci in which 5hmC expression increased and 3481 genes in which 5hmC content decreased In the analysis of the distribution trend of DhMRs,it was found that DhMRs are enriched in genes such as translation regions,promoters and exons,but are rare in intergenic regions,similar to the previous results.However,in the analysis of GO pathways,the function of DhMRs is different from the first two groups of DhMRs,especially in the DhMRs sites where 5hmC declines,mainly related to the signal transduction pathways involved in splicing and nucleoside regulation and GTPase-mediated regulation.In the DhMRs sites with increased expression of 5hmC,Ras-related signaling pathways are mainly related to splice variants,etc.6.DhMRs have significant coincidence with the identified gastric cancer superenhancer,5hmC is closely related to gastric cancer.ConclusionIn summary,changes in epigenetics played an important role in the development of gastric malignancies.Our results show that the reduction of 5hmC expression level is closely related to gastric tumors and is specific in gastric cancer.In the specific hydroxymethylation region,we found that the diferential sites are mostly located inside the tumor genes,and 5hmC may be involved in tumorigenesis.By analyzing DhMRs and related signaling pathways,DhMRs(the loss of 5hmC and the increase of 5hmC)are enriched in genes involved in many selective pathways.In the phantom analysis,we found that the change of 5hmC in the occurrence and development of gastric cancer may lead to changes in the binding of HIF1 and Myc to DNA.In the analysis of tumor progression,we found that the reduction of 5hmC content was more significantly related to gastric tumor progression,and it was speculated that the loss of 5hmC may cause RAS gene imbalance and lead to gastric cancer progression.In addition,we observed a clear overlap between DhMR and the previously identified super enhancers.These data together indicate that the change of 5hmC may potentially promote the occurrence and development of gastric cancer.Studying gastric cancer and 5hmC can provide important guidance for early diagnosis,treatment and prognosis evaluation. |
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