| Objective(s): Cell-free DNA(cf DNA),also known as plasma-free DNA,is a double-stranded DNA fragment that is present in human plasma,urine,and cerebrospinal fluid and is free of cells.Recently,cf DNA has been applied in many diseases as a novel marker for noninvasive screening and diagnosis,disease monitoring and efficacy evaluation.Eukaryotic translation initiation factor 2C1(EIF2C1)is often used as an internal reference for digital polynucleotide chain reaction(PCR).This study aims to establish a method for quantitative detection of plasma EIF2C1 based on real-time fluorescent quantitative PCR(RT-qPCR)technology,which can effectively reflect the total amount of circulating free DNA,and then further evaluate the application value of EIF2C1 in the diagnosis and prognosis of primary hepatocellular carcinoma(PHC).Method(s):(1)Establishment of EIF2C1 quantitative detection method: Two pairs of primers and probes for EIF2C1 were designed,and the human genome DNA standard material was used as the standard substance.Plasma of 10 patients with PHC was collected,cf DNA in plasma was extracted by centrifugal column method,and EIF2C1 level in plasma was detected by RT-qPCR technique.The consistency of the results of two primers and probes was evaluated.(2)A total of 116 patients diagnosed with PHC and 90 healthy subjects admitted to the Affiliated Hospital of Guilin Medical University between January 2022 and November 2022 were enrolled in the study.Venous blood(2m L)was acquired in the early morning of the second day of hospitalization,and plasma EIF2C1 level was quantitatively detected by the above method.The general clinical data of patients were collected and statistically analyzed using SPSS26.0 software.Result(s):(1)A method for detection of plasma EIF2C1 based on RTqPCR was established,and the results of the two primers and probes were consistent,indicating that the detection method was reliable.(2)The EIF2C1 content in the PHC group was significantly higher than that in the control group(P<0.001).The plasma EIF2C1 content of patients with PHC was negatively correlated with indirect bilirubin(I-BIL),albumin/ globulin(A/G),prealbumin(PA),albumin(ALB),and cholinesterase(CHE).The plasma EIF2C1 content of patients with PHC was positively correlated with alkaline phosphatase(ALP),γ-glutamyl transpeptidase(γ-GT),and alpha-fetoprotein(AFP).(3)The level of EIF2C1 in PHC patients with clinical stage I was significantly lower than that in PHC patients with stage III(P<0.05)and stage IV(P<0.01),and the level of EIF2C1 in PHC patients with cirrhosis was higher than that in patients without cirrhosis.The lower the Child-Pugh grade was,the lower the EIF2C1 level was(P<0.05).The level of EIF2C1 in PHC patients with tumor metastasis was significantly higher than that without metastasis(P<0.001).(4)EIF2C1could be used to independently diagnose PHC(EIF2C1-1 AUC: 0.883,sensitivity 94.0%,specificity 73.3%,cut-off value: 6617.0 copies/μL,positive rate 81.95%,negative rate 90.41%;EIF2C1-2 AUC: 0.887,sensitivity 94.0%,specificity 71.1%,cut-off value:7633.0 copies/μL,positive rate 80.74%,negative rate 90.14%).The combined detection of EIF2C1 with AFP,carcinoembryonic antigen(CEA),and carbohydrate antigen 125(CA125)improved the diagnostic efficiency(EIF2C1-1+AFP+CEA+CA125: sensitivity 100%,specificity 89.5%,AUC: 0.987;EIF2C1-2+AFP+CEA+CA125: sensitivity 84.0%,specific-ity 100%,AUC: 0.977).(5)The indicators with significant correlation in the univariate analysis were included in the binary logistic regression analysis model to assess the risk of PHC.The results showed that the risk of PHC was increased with the increase of age,sex,total bilirubin(T-BIL),globulin(GLO),alanine aminotransferase(ALT),aspartate transferase(AST),EIF2C1,AFP,and CEA(EIF2C1-1 OR: 7.74;EIF2C1-2 OR: 6.42);The indicators of significant correlation in the univariate analysis were included in the binary logistic regression analysis model to evaluate the risk of tumor metastasis,and the results showed that the increase of EIF2C1 was indicative of the increased risk of tumor metastasis.(EIF2C1-1 OR: 4.46;EIF2C1-2 OR: 8.46).Conclusion(s):(1)Our study establishes a method for quantitative detection of EIF2C1 based on RT-qPCR technology.The detection method is simple,stable,and reliable,which is conducive to further laboratory research and promotion in hospitals,and provides a new strategy for clinical diagnosis.(2)The level of EIF2C1 is significantly increased in patients with PHC,and a high level of EIF2C1 indicates a higher risk of clinical stage,liver function grade,cirrhosis,and tumor metastasis.EIF2C1 combined with tumor markers(AFP/CEA/CA125)can significantly improve the diagnosis rate of PHC.EIF2C1 has potential application value in predicting the risk of metastasis. |
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