Study On The Correlation Between MRNA M6A Methylation Differences And The Risk Of Gastrointestinal Stromal Tumors By Using Apparent Transcriptome Chip Technology

Part ? Exploring the Clinical Characteristics and Prognosis of 751 Patients with very Low / Low Risk Gastrointestinal Stromal Tumors Background and PurposeAccording to the consensus standard(2008)of the National Institutes of Health(NIH),GIST with a diameter of ? 5 cm and a mitotic counting of ? 5/50 HPF is considered as a very low / low risk GIST.For these patients,the rate of recurrence and distant metastasis are extremely low,and adjuvant imatinib treatment after surgery is not necessary.Their treatment plan is observation and periodic imaging evaluation.However,some very low / low risk GIST with recurrence / metastasis have been found clinically.This makes us question the existing indicators for evaluating GIST risk factors.The purpose of this article is to explore the clinical features of very low / low risk GIST.MethodsFrom January 2012 to October 2018,751 very low / low risk GIST patients were selected in our hospital.Recording and analyzing the basic information,clinicopathological characteristics and clinical results of these patients.ResultsMost GIST(594/751)occurs in the stomach.Endoscopic submucosal dissection(ESD)was excised in 361 cases,laparoscopic surgery in 198 cases and open surgery in 192 cases.The overall resection rate is 100%.Among them,there were 516 cases(69%)of low-risk GIST and 235 cases(31%)of high-risk GIST.At a median follow-up of 38(7 to 94)months,534 patients were found to have 4 relapses and 1 distant metastasis.ConclusionVery low / low-risk GIST shows extremely low rates of recurrence and distant metastasis.However,there are still 4 relapses and 1 distant metastasis case in our study.According to the existing NIH standards to divide the risk of stromal tumors as certain limitations.Part ? m6 A Apparent Transcriptome Microarray for Screening RNA Related to the Risk of Gastrointestinal Stromal Tumor and Using Real-time Quantitative PCR to Validate PurposeBy comparing the difference of the transcriptome m6 A methylation modification levels of high-risk and low-risk stromal tumor tissue samples collected in clinical work.To explore the effect of epigenetics on the difference in the risk of gastrointestinal stromal tumors.Focus on the difference between m6 A methylation level and methylation quantity of m RNA,and then finding new tumor markers of gastrointestinal stromal tumors,which are providing a theoretical basis for the gastrointestinal stromal tumor of epigenetics and providing a new indicators for the judgment of the risk of gastrointestinal stromal tumors.MethodsCollecting the tumor specimens of patients with gastrointestinal stromal tumors who underwent surgical treatment in the First Affiliated Hospital of Zhengzhou University from November 2015 to April 2019,and the diagnosis and risk of the tissue removed from the surgery were divided into the diseases of the First Affiliated Hospital of Zhengzhou University based on pathological diagnosis of science.After excluding basic diseases,age,gender and other interference factors,10 samples of stromal tumors(5 cases of high-risk stromal tumors and 5 cases of low-risk stromal tumors)were selected from them,and the transcriptome m6 A methylation was detected.In short,after extracting the total RNA,immunoprecipitation of total RNA is precipitated by using specific anti-m6 A antibodies,and then the human apparent transcriptome chip of Arraystar is used to detect the m6 A methylation level and methylation amount of the sample.The difference between m6 A RNA methylation level and methylation amount between high-risk and low-risk stromal tumors is filtered by setting the fold change(FC)and the thresholds of statistical significance(p-value).The m RNA modified by differential m6 A in the obtained results was analyzed for GO function enrichment and KEGG pathway,and the tumor-related m RNA was screened out.Finally,the gastrointestinal stromal tumor tissues were detected by real-time quantitative PCR and western blotting to verify the m RNA expression differences.Results1.The results of the total RNA quality identification showed that the overall RNA quality was reliable,and the tissues of all the samples were not degraded or contaminated by impurities,and subsequent experiments such as microarray chips can be performed.Arraystar's Human m RNA & lnc RNA Epitranscriptomic Microarray(8x60K,Arraystar)chip was used to detect m6 A methylation levels and methylation levels of m RNA in samples from high-risk and low-risk groups of gastrointestinal stromal tumors.The results showed that in terms of methylation level,a total of 168 up-regulated m RNAs and 46 down-regulated m RNAs were screened;in terms of methylation amount,a total of 4492 up-regulated m RNAs and 201 down-regulated m RNAs were screened.2.After screening under the above conditions,the resulting m RNAs with increased levels of m6 A methylation and methylation were subjected to GO functional enrichment analysis and KEGG pathway analysis.The results showed that in terms of methylation level,the results of GO functional enrichment analysis mainly concentrated on the positive regulation of Wnt signaling pathway,positive regulation of gene expression,cell surface,extracellular matrix,protein binding and heparin binding and other cell functions or structure;KEGG pathway analysis results are mainly enriched in PI3K-Akt signaling pathway,Rap1 signaling pathway and other pathways.In terms of methylation amount,the results of GO functional enrichment analysis are mainly enriched in cell functions or structures such as cell division,cell proliferation,cytoplasm,nucleus,ATP binding,and Actin binding;KEGG pathway analysis results are mainly enriched in PI3 K Akt signaling pathway,Focal adhesion signaling pathway and other pathways.Among them,the PI3K-Akt signaling pathway is the most significant pathway in the analysis of KEGG pathway,both in terms of methylation level and methylation amount.Moreover,molecules such as VEGFA?INSR and FN1 are involved in these pathways.3.Qrt-pcr and WB results showed the relative level of expression of VEGFA,INSR and FN1 m RNA in high-risk stromal tumors were higher than those in low-risk stromal tumors(p <0.05).Conclusion1.Differential methylation modification of PI3K-AKT pathway-related molecules is likely to be a factor affecting the risk of stromal tumors.2.The m6 A methylation modification degree such as VEGFA,INSR and FN1 of m RNA in high-risk mesenchymal tumors was higher than that in high-risk mesenchymal tumors.3.The increased expression levels of m RNA of VEGFA,INSR and FN1 may be related to the higher risk rating of stromal tumors.4.The increased expression levels of m RNA of VEGFA,INSR and FN1 may be due to the increased level of m6 A methylation.