| BackgroundLiver cancer is the sixth most common and the third most deadly malignancy worldwide in 2020.with about 905,677 new cases and 830.180 deaths.China is one of the most high-risk areas of HCC.Liver cancer starts insidiously,develops rapidly,and its heterogeneity leads to a high degree of malignancy and diverse biological behaviors;when detected,it is often at an advanced stage of the disease,making clinical treatment intervention more difficult.Approximately 85%-90%of primary liver cancers are HCC.Hepatitis B virus(HBV)infection is the most common causative factor of HCC.Liver tissue puncture biopsy is the most accurate approach to diagnose HCC,but puncture biopsy is an invasive operation and cannot be used as a routine test in clinical practice.Currently,alpha-fetoprotein(AFP)is the most commonly used serological biomarker for screening HCC in clinical practice,which is simple and noninvasive but has poor sensitivity and specificity.Therefore,it is badly needed in clinical practice for biomarkers which can be used for screening and early diagnosis of HCC.Wntl-inducible signaling pathway protein 1(WISP1),a member of the CCN growth factor family,regulates cell multiplication,migration,survival and adherence,and plays a vital role in the formation and prognosis of many tumors.Aberrant WISP1 expression is associated with the formation of hepatocellular carcinoma.DNA methylation is by far one of the most common epigenetic alterations in HCC.We hypothesize that DNA methylation is involved in the regulation of WISP1 expression.AimsDetecting the WISP1 promoter methylation status and the relative expression of WISP1 in patients with HCC,chronic hepatitis B(CHB)and healthy controls(HCs),thus exploring the potential clinical value of WISP1 methylation levels for the diagnosis of HCC.MethodsThe study enrolled 251 participants,including 123 participants with HCC,90 participants with CHB and 38 HCs.Peripheral blood was acquired from all subjects,peripheral blood mononuclear cells(PBMCs)and plasma were extracted from the whole blood.DNA and RNA were obtained from the PBMCs.Bisulfite conversion of DNA were performed,and RNA were reverse transcribed into cDNA.The method used in this study to detect the methylation status of WISP1 promoter in all participants was methylation-specific polymerase chain reaction(MSP),and the method used to determine the mRNA expression level of WISP1 was real-time quantitative PCR(RT-qPCR),and the method used to detect the expression of plasma soluble WISP1 was enzyme-linked immunosorbent assay(ELISA).Results1.Thirty-five of 123(28.5%)patients with HCC showed WISP1 gene promoter methylation,while 55 of 90(61.1%)patients with CHB and 36 of 38(94.7%)HCs exhibited WISP1 promoter methylation.The methylation frequency of WISP1 in patients with HCC was predominantly lower than that of patients with CHB and HCs(P<0.001).At the same time,the methylation frequency of WISP1 in the CHB group was lower than that in the HCs group(P<0.001).2.In patients with HBV-associated HCC,WISP1 methylated patients have a lower AFP level than unmethylated patients(P=0.001).Methylation frequency in TNM stage Ⅲ/Ⅳ patients(17.3%)was lower than in TNM stage Ⅰ/Ⅱ(36.6%,P=0.019).Meanwhile,the methylation frequency in tumor size≥ 5 cm patients(16.7%)was lower than in tumor size<5 cm(39.7%,P=0.005).3.The mRNA levels of WISP1 in the HBV-associated HCC were significantly greater than those in the CHB(P<0.001),and the mRNA levels in the CHB were also higher than those in the HCs(P=0.043).In the HCC group,mRNA levels were obviously lower in methylated patients than in those unmethylated(P<0.001).In addition,the mRNA levels were higher in HBeAg-positive patients than in HBeAg-negative patients(P=0.029)4.Plasma soluble WISP1 concentrations were lower in patients with HCC than in patients with CHB(P=0.026)and HCs(P<0.001).Plasma WISP1 levels were obviously higher in the methylated patients than in the unmethylated patients(P=0.013).Meanwhile,the level of plasma WISP1 showed significance with TNM stage(P=0.013),vascular invasion(P=0.008)and tumor size(P=0.019).The concentration of plasma WISP1 was negatively correlated with WISP1 mRNA expression(r=-0.171,P=0.015)and AFP level(r=-0.148,P=0.033).5.Abnormal WISP1 gene methylation presented a sensitivity of 71.45%and specificity of 57.95%in distinguishing patients with HCC from patients with CHB,and AFP showed a sensitivity of 53.39%and specificity of 82.56%.In addition,the combination of WISP1 methylation and serum AFP levels displayed a sensitivity of 71.19%and specificity of 67.44%.The area under the receiver operating characteristic curve(AUC)of WISP1 promoter methylation and AFP levels was 0.647 and 0.667,and there was no statistical difference between them.The AUC of the combined determination(0.736)was markedly greater than that of serum AFP alone(P=0.027)Conclusion1.Compared with patients with CHB,the methylation frequency of WISP1 gene promoter in PBMCs of patients with HBV-associated HCC was significantly lower,while the expression level of mRNA was significantly higher and the plasma WISP1 level was significantly lower,indicating that the promoter region of WISP1 in patients with HCC was hypermethylated and the hypomethylation status of the promoter might negatively regulate the expression of mRNA.The inconsistency of mRNA expression and protein expression may be regulated by other biological processes2.In patients with HBV-related HCC,there were significant differences in TNM stage and tumor size between methylated and unmethylated patients,while plasma WISP1 levels correlated with indicators such as TNM stage,vascular invasion and tumor size.Indicators such as TNM stage,tumor size,and vascular invasion are vital predictors of progression and prognosis of HCC.Therefore,we speculate that the methylation status of WISP1 and plasma WISP1 levels may be associated with the progression and prognosis of HBV-related HCC.3.The combination of methylation WISP1 gene promoter with AFP has potential diagnostic value in HBV-associated HCC,suggesting that WISP1 methylation status combined with AFP may serve as a noninvasive biomarker for diagnosis of hepatocellular carcinoma,and it will provide a new direction for the diagnosis and prognosis of HCC. |
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