Clinical Diagnostic Value Of Human Epididymis Protein 4(HE4) And 5-methylcytosine(m~5C) Modification Of Immune Cells In Peripheral Blood In Colorectal Cancer

ObjectiveThis study investigates the expression level of human epididymis protein 4(HE4)in the peripheral blood of colorectal cancer(CRC)patients and its diagnostic value.This study will also explore the level of m5C methylation in immune cells of CRC patients and its clinical application value.MethodsPart Ⅰ:259 patients from the Seventh Affiliated Hospital of Sun Yat-sen University were enrolled in this study,including 146 patients with benign intestinal lesions(the benign control group),and 113 patients with CRC(the malignant group.).169 healthy people(the healthy group)were also included in this study.Serum levels of carcinoembryonic antigen(CEA),carcinoembryonic antigen 19-9(CA19-9)and HE4 were detected,and the receiver operating characteristic curve(ROC curve)was drawn to evaluate the diagnostic value.Part Ⅱ:53 healthy people and 92 untreated colorectal cancer patients in Zhongshan People’s Hospital were collected as the training set.Thirty healthy people and 62 colorectal cancer patients in Sun Yat-Sen University Cancer Center were collected as the validation set.MC38 murine CRC model and AOM/DSS induced CRC model were established in C57BL/6 mice.The level of m5C methylation in total RNA of immune cells were detected.The clinical value was assessed,and independent risk factors were established.Gene set variation analysis(GSVA)was performed based on the GEO database to obtain the critical factors in the methylation modification of m5C.qRT-PCR was used to detect the expression of the above key factors.Again,GSVA evaluation was performed based on GSE10715 to establish the primary immune cell types modified by m5C in peripheral blood.ResultsPart 1:Diagnostic value of serum HE4 in colorectal cancer.(1)The serum levels of CEA,CA19-9 and HE4 in the malignant group were significantly higher than those in both healthy and benign control groups,which were positively correlated with the incidence of colorectal cancer.(2)The area under the curve(AUC)of serum markers CEA,CA]9-9,HE4 and combination test were 0.773,0.622,0.767 and 0.816,respectively.Part Ⅱ:Clinical value of m5C methylation level in peripheral blood immune cells for colorectal cancer.(1)The m5C modification level in the total RNA of immune cells in the peripheral blood of CRC patients was significantly higher than healthy people.The m5C level in MC38 Syngeneic mice models and primary CRC mice models with the chemical inducer AOM/DSS were markedly higher than in C57BL/6 controls.(2)The methylation level of m5C in patients with early colorectal cancer was also significantly higher than that in healthy people.And it increased with the clinical stage and metastasis.The AUC in the training and validation sets were 0.697 and 0.795,respectively.(3)The level of m5C methylation of RNA in immune cells in peripheral blood of patients with colorectal cancer after treatment was significantly decreased.(4)Both in the training and validation sets,the AUC of the ROC curve of the m5C methylation of RNA in peripheral blood immune cells was 0.888 and 0.909,and the sensitivity was 92.40%and 93.50%.(5)In the training set,the AUC of serum CEA,CA125 and CA19-9 in diagnosing colorectal cancer were 0.739,0.629 and 0.669,respectively.The AUC of m5C+CEA+CA125+CA19-9 combined detection was 0.937.In the validation set,the AUC of serum CEA and CA19-9 for diagnosing colorectal cancer was 0.622,0.637,and the AUC of m5C+CEA+CA19-9 combined detection for diagnosing colorectal cancer was 0.926.(6)Multivariate logistic regression analysis showed that serum CEA and the level of m5C methylation of total RNA immune cells in peripheral blood were independent risk factors for the diagnosis of colorectal cancer.(7)The level of NSUN5 and YBX1 in colorectal cancer patients were significantly higher than those in healthy people.NSUN5 and YBX1 level were significantly correlated with the methylation level of total RNA m5C and the infiltration of monocytes.Conclusion(1)HE4 has potential diagnostic value for colorectal cancer.(2)The m5C methylation level of total RNA in peripheral blood immune cells of patients with colorectal cancer was significantly increased,correlated with clinical analysis and metastasis,and may act as a biomarker for diagnosis and clinical monitoring of colorectal cancer.(3)NSUN5 and YBX1 are the key m5C-modified regulators in the immune cells of CRC patients.(4)Monocytes are the major m5C methylation-modified immune cell type in the blood of colorectal cancer patients.