| BackgroundMajority(70%to 90%)of primary liver cancer in the world is hepatocellular carcinoma(HCC),ranking fifth in the world’s most common cancers and ranking second among cancer-related causes of death,mainly related to chronic infection with hepatitis B virus(HBV)or hepatitis C virus(HCV).Chronic infection of HBV or HCV is more prevalent in low and middle income countries.It is estimated that 50%of liver cancer cases and deaths worldwide occur in China.The number of liver cancer deaths in China in 2005 was 339,308,including 246,808 in men and 92,500 in women,and the number of incident liver cancer cases was estimated to be 370,236,including 274,562 in men and 95,674 in women.Although the level of diagnosis and treatment of HCC is higher than before,more than half of HCC patients die within 12 months after diagnosis,and less than 6%HCC patients have an average survival rate of 5 years.Therefore,early diagnosis and treatment of HCC is necessary.Currently,the main means of diagnosis and screening of HCC mainly include alpha-fetoprotein(AFP)levels,ultrasound(US),computed tomography(CT)and magnetic resonance imaging(MRI).AFP is currently the most commonly used serological screening indicator in the clinic.However,studies have shown that AFP lacks sufficient sensitivity and specificity.AFP can be elevated during pregnancy,and abnonnal elevations have been reported in gonadal tumors(germ cells and non-germ cells)and other malignancies.Some studies have reported a rise of AFP in patients with chronic liver disease without HCC and patients with acute or chronic viral hepatitis.Although AFP greater than 400 ng/ml is used for the diagnosis of HCC,this high level of AFP appears only in a small number of patients.The utility of AFP in differentiating HCC from benign liver disease is also considered to be limited by the rate of false negatives and high false positive rates.Because HCC has different ways of reflection on ultrasound,14 studies evaluating ultrasound have reported low sensitivity to ultrasound.CT has radiation exposure and relatively low soft tissue contrast.MRI is more time consuming,more prone to artifacts,and requires more expertise to analyze and interpret images,so MRI has lower usability.The gold standard for liver cancer diagnosis is liver biopsy and pathological analysis,but liver biopsy is an invasive test with certain traumaticity.Therefore,it is necessary to find new non-invasive biomarkers to detect hepatocellular carcinoma at earlier stage.Natural killer cell receptor group 2D(NKG2D)is an important activating receptor in the innate immune system.Its killing effect on target cells is achieved by identifying ligands on the surface of target cells,transmitting activation signals and activating the immune system.NKG2D plays an important role in the immune regulation of tumors.It initiates innate immune surveillance and clearance in the early stages of tumorigenesis.Studies have shown that in the peripheral blood of tumor patients,the expression level of NKG2D receptor on the surface of NK cells is significantly decreased.It is now widely accepted that cancer is caused by a combination of genetic and epigenetic damage or dysfunction.Although the underlying cause of cancer is still difficult to define,it is clear that environmental factors can alter the epigenome and ultimately increase the risk of cancer.DNA methylation is the most common form of epigenetic alteration,and we speculate that it is involved in the regulation of the NKG2D gene.ObjectiveDetecting the methylation levels of NKG2D promoter in peripheral blood mononuclear cells(PBMCs)of HCC patients,chronic hepatitis B(CHB)patients and healthy controls(HCs),so as to explore the diagnostic value of NKG2D promoter methylation in HCC.MethodsThis study included 101 HCC patients,56 CHB patients,and 32 HCs.Peripheral blood of the study subject was obtained and PBMCs were isolated from whole blood.DNA and RNA were extracted from PBMCs.DNA is methylated and RNA is reverse transcribed.We investigated the methylation status of the NKG2D promoter by methylation-specific polymerase chain reaction(MSP)and the mRNA expression level was examined by real-time quantitative polymerase chain reaction(RT-qPCR).Results1.Comparison of basic clinical features of three groups:There were no significant differences in age and gender between HCC patients,CHB patients and HCs(P>0.05).There were significant differences in ALT,AST,TBIL,ALB,and Cr(P<0.001).There were no significant differences in HBsAg,HBV DNA,INR,PTA and AFP between HCC patients and CHB patients(P>0.05),and the difference of HBeAg was statistically significant(P<0.001).2.The methylation frequency of NKG2D promoter in HCC patients(63/101,62.4%)was significantly higher than that in CHB patients(10/56,17.9%;X2=28.701,P<0.001)and HCs(0/32,0.0%;X2=37.925,P<0.001).The methylation frequency of NKG2D promoter in CHB patients and HCs was also significantly different(X2=6.447,P=0.011).3.In HCC patients,TNM stage of the NKG2D promoter methylation group and the unmethylation group was statistically different(X2=5.477,P=0.019),and there were no significant differences between the two groups in age(Z=-0.232,P=0.817),gender(X2=0.926,P=0.336),smoking(X2=0.404,P=0.525),alcohol(X2=0.701,P=0.402),HBsAg(Z=-1.609,P=0.108),HBeAg(X2=1.311,P=0.252),HBV DNA(X2=0.029,P=0.865),ALT(Z=-1.459,P=0.145),AST(Z=-0.519,P=0.604),TBIL(Z=-0.638,P=0.524),ALB(Z=-1.911,P=0.056),INR(Z=-0.224,P=0.822),PTA(Z=-0.004,P=0.997),Cr(Z=-1.893,P=0.058),AFP(Z=-0.175,P=0.861),tumor size(X2=1.473,P=0.225),tumor number(X2=0.019,P=0.890),vascular invasion(X2=0.901,P=0.342),CTP stage(X2=0.936,P=0.626),and histological grade(X2=3.520,P=0.172).In addition,multivariate logistic regression analysis was performed by using age,gender,HBsAg,HBeAg,HBV DNA,ALT,AST,TBIL,ALB,INR,PTA,Cr,AFP,number of tumors,vascular invasion,tumor size,TNM staging as independent variables,NKG2D promoter methylation as a dependent variable.The results showed that TNM stage increased NKG2D promoter methylation.4.NKG2D mRNA levels were significantly lower in HCC patients than in CHB patients(Z=-5.015,P<0.001)and HCs(Z=-8.296,P<0.001).The difference in NKG2D mRNA levels between CHB patients and HCs group was also statistically significant(Z=-6.900,P<0.001).In HCC patients,NKG2D mRNA levels were lower in the methylation group than that in the unmethylated group(Z=-2.236,P=0.025).The methylation status of NKG2D promoter was negatively correlated with the NKG2D mRNA levels in HCC patients(r=-0.224,P=0.025).In HCC patients,there were no significant difference in NKG2D mRNA levels between HBeAg(+)group and HBeAg(-)group(Z=-1.472,P=0.141),and NKG2D mRNA levels in HBV DNA(+)group were lower than that in HBV DNA(-)group(Z=-2.243,P=0.025),NKG2D mRNA levels in TNM staging(III/IV)group were lower than that in TNM staging(I/II)group(Z=-3.404,P=0.001)and NKG2D mRNA levels in vascular invasion(+)group were lower than that in vascular invasion(-)group(Z=-2.302,P=0.021).In HCC patients,NKG2D mRNA levels were correlated with AFP(r=-0.294,P=0.003),TBIL(r=-0.207,P=0.037),AST(r=-0.224,P=0.024),TNM staging(r=-0.340,P<0.001),HBV DNA(r=-0.224,P=0.024)and vascular invasion(r=-0.230,P=0.021).NKG2D mRNA levels were not associated with ALT(r=-0.164,P=0.102),HBsAg(r=0.015,P=0.884),INR(r=0.121,P=0.228),PTA(r=0.127,P=0.204),ALB(r=0.147,P=0.141).5.The sensitivity of NKG2D promoter methylation to distinguish HCC and CHB was 62.4%,the specificity was 82.1%,the positive predictive value was 86.3%,and the negative predictive value was 54.8%.The sensitivity of AFP in the diagnosis of HCC was 48.5%,the specificity was 50.0%,the positive predictive value was 63.6%and the negative predictive value was 35.0%.When combined AFP and NKG2D promoter methylation,the sensitivity of distinguishing HCC and CHB was 81.2%,the specificity was 48.2%,the positive predictive value was 73.9%,and the negative predictive value was 58.7%.Compared with AFP alone,the combination of AFP and NKG2D promoter methylation has higher sensitivity(X2=23.651,P<0.001)and negative predictive value(X2=6.667,P=0.010).The difference was not statistically significant in specificity(X2=0.036,P=0.850)and positive predictive value(X2=2.255,P=0.133).When AFP was greater or equal to 20 ng/ml,the HCC detection rate in the NKG2D methylation group(30/39,76.9%)was significantly higher than that in the unmethylated group(19/38,50.0%;X2=6.029,P=0.014).When AFP was less than 20 ng/ml,the detection rate of HCC in the methylation group(33/34,97.1%)was also higher than that in the unmethylated group(19/46,41.3%;X2=26.713,P<0.001).The area under the receiver operating characteristic curves(AUC)for NKG2D promoter methylation for distinguishing HCC from CHB was 0.723(SE 0.0354,95%CI 0.646-0.791),which was significantly higher than the AUC level of AFP(AUC 0.569,SE 0.0477,95%CI 0.488-0.648).The difference was statistically significant(P=0.0037).The AUC of the combination of the two markers(AUC 0.647,SE 0.0389,95%CI 0.567-0.722)was also higher than that of AFP alone(AUC 0.569,SE 0.0477,95%CI 0.488-0.648;P=0.0141).Conclusions1.There was abnormal methylation of NKG2D promoter in HCC patients,and the methylation rate was higher than that of CHB patients and HCs.Its mRNA expression level was lower than that of CHB patients and HCs,suggesting that hypermethylation of NKG2D promoter may lead to down-regulation of gene expression.2.In HCC patients,there was a statistically significant difference in TNM staging between the NKG2D promoter methylation group and the unmethylated group,and TNM staging increased NKG2D promoter methylation.TNM staging is an important indicator to evaluate the progression of HCC,suggesting that NKG2D promoter methylation may be related to HCC progression.HCC mRNA expression levels were significantly negatively correlated with AFP,TBIL,AST,HBV DNA,TNM stage and vascular invasion.These indicators can reflect the severity of the lesion,suggesting that methylation of the NKG2D promoter may be related to the severity of the lesion.3.NKG2D promoter methylation had a good predictive value in HCC,suggesting that NKG2D promoter methylation can be used as a non-invasive marker for detecting HCC at earlier stage and provide new ideas for the diagnosis and treatment of HCC. |
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