Exploration And Optimization Of Cell-free DNA Methylation Detection Technology For Colorectal Cancer Early Screening

Colorectal cancer(CRC)is a malignant gastrointestinal tract tumor.Colorectal cancer is the third most common cancer and the fourth most common cancer cause of death globally.The average 5-year survival rate of metastatic CRC is less than 10%,but if cancer is detected at early stage,it can increase the average 5-year survival rate to 90%.Therefore,early screening is a significant method to prevent CRC.Current early screening methods for colorectal cancer are limited by invasiveness and low sensitivity,and they are currently only offered to a small proportion of the general population.Therefore,to develop non-invasive testing technology could be a promising alternative to early screening for CRC.Cell-free DNA(cf DNA)may enable a noninvasive ‘liquid biopsy’ for early screening of CRC.Compared with the current methods,it has some obvious advantages,such as minimally invasive,dynamic monitoring,and not affected by intra-tumor heterogeneity.DNA methylation plays a key role in the control of gene activity and nuclear structure,is one of a series of epigenetic modification can regulate gene expression.In tumor cells,the promoter region or the first exon region of some tumor suppressor genes usually becomes clusters of guanine and cytosine,called Cp G islands.If Cp G islands of the tumor suppressor genes are hypermethylated,the expression of these genes are inhibited,resulting in tumorigenesis.Aberrant DNA methylation is closely related to the occurrence and development of tumors.Detection of DNA methylation alters also provides a new way for the diagnosis and treatment of cancer.Studies have shown that circulating tumor DNA(ct DNA)as methylation biomarkers have great application prospects in early screening,diagnosis and prognosis surveillance.In this study,we use the bisulfite conversion technology and multi-reaction real-time fluorescent quantitative PCR technology to explore and optimize the accuracy and efficiency of cell-free DNA methylation detection.To study aberrant DNA methylation in CRC,we analyzed target genes with differential methylation between CRC samples and normal samples by using the gene chip sequencing technology.We detected DNA methylation level in plasma cf DNA from 65 CRC patients and 27 healthy controls.We analyzed the correlation between the DNA methylation level of the target genes in plasma cf DNA and stage,age,and gender of CRC patients and showed that the methylation level of the target gene is significantly correlated with tumor stage and age.We constructed a diagnostic prediction model and evaluated the diagnostic feasibility of SDC2,BMP3,and NDRG4 genes as biomarkers for CRC early screening by plotting ROC curves.The SDC2 gene was evaluated as the optimal screening target gene.In summary,we explored the cell-free DNA methylation detection technology,and the goal of improving detection accuracy and reducing detection cost was achieved.We used methylation detection technology to study the blood samples of CRC patients and healthy controls,and obtained the alteration of target genes methylation in CRC through differential methylation analysis and correlation analysis,and initially found out CRC early screening markers for the Chinese population.This study will provide a certain theoretical and experimental basis for illuminating the molecular mechanism of aberrant methylation in CRC,and provide a certain experimental basis for optimizing CRC early screening technology.