Methylation Of DKK3 And IHIT5 As A Potential Biomarker For Hepatocellular Carcinoma

BackgroundHCC is worldwide the fifth most common cancer in men and the seventh one in women,the third most common cause of cancer-related deaths worldwide(1).Most HCC cases(<80%)are attributable to hepatitis B virus(HBV)or hepatitis C virus(HCV)infections,explaining the distinct geographic distribution of HCC with most cases found in developing countries with endemic infections.Finding ways to detec hepatocellular carcinoma at an earlier stage making patients can get the surgical resection and other radical treatment opportunities is a hot issue.Currently,the combination of AFP which is used as a well-known biomarker for HCC and ultrasonography is widely used for HCC screening and surveillance.But several studies showed that AFP have low sensitivity which is only 22%to 60%and poor diagnostic yield at the early stage of HCC.(4-5)Liver biopsy is gold standard to diagnose HCC,but is too traumatic.New noninvasive biomarkers are needed to detect hepatocellular carcinoma at an earlier stage and to individualize treatment strategies.DKK3 is a member of the Dickkopf family,which is a group of secreted glycoproteins known to inhibit the Wnt signaling pathway.Several studies showed that down-regulation or silencing of DKK3 is associated with promoter CpG methylation in chronic lymphocytic leukemia,myelodysplastic syndrome,AML and ALL[14-19].There was a study already demonsterated that the aberrant promoter methylation of DKK-3 in HCC tissue may be an important mechanism in HCC,but information on DKK3 promoter methylation in HCC is still lacking.ITIH5 is is a novel member of the inter-a-trypsin inhibitor family which can be linked with hyaluronic acid to stabilize the ECM。It is identified as a tumor suppressor.Serveral studies showed that aberrant ITIH5 methylation results in the loss of ITIH5 incolon,lung(28),gastric(29)and cervical cancer(30).But the methylation status of ITIH5 promotor and first exon in PBMCs of HCC patients is still unclear.This study aimed to investigate the possibility of DKK3 promoter and ITIH5 promoter and first exon methylation in PBMCs as a new potenial biomarker in HCC.Methods Methylation status of DKK3 and ITIH5 in PBMCs was examined in a total of 201 subjects,including 113 patients with HCC、60 patients with CHB and 30 healthy controls using methylation-specific polymerase chain reaction(MSP)and their mRNA levels was detected by quantitative real-time polymerase chain reaction after DNA、RNA extraction from PBMCs and cDNA Synthesis.Results1.Hypermethylation of the DKK3 and ITIH5 in PBMCs both occurred significantly more frequent in HCC than CHB(p=0.000,p=0.000)and HCs(p=0.000,p=0.000),respectively.No significant difference of methylation frequency of DKK3 and ITIH5 was observed between CHB patients and HCs(P=0.878 and P=0.750).2.The mRNA concentrations of DKK3 and ITIH5 in HCC were significantly lower than CHB(P=0.0020,P<0.0001)and HCs(P<0.0001,P<0.0001).Significant differences of the DKK3(P=0.0011)and ITIH5(P=0.0003)mRNA expressions were found in CHB group than in HCs.3.Hypermethylation of DKK3 promoter was found in patients with larger tumor size(>3 cm)compared with smaller tumor size(≤3cm)(P=0.020)and DKK3 mRNA was related to tumor size(P=0.0132).ITIH5 methylation frequency was elevated in HCC patients with vascular invasion and advanced TNM stages(Ⅲ-Ⅳ)compared with those without vascular invasion and early stage(Ⅰ-Ⅱ)(P=0.012,P-0.025)and ITIH5 mRNA in HCC patients with vascular invasion and advanced TNM stages(Ⅲ-Ⅳ)was lower than those without vascular invasion(P=0.0090)and early stage(Ⅰ-Ⅱ)(P=0.0203).4.To discriminate HCC from CHB,DKK3 promoter and ITIH5 promoter、first exon methylation showed a sensitivity of 73.45%and 75.22%.Combined ITIH5 and DKK3 methylation in PBMCs showed a higher sensitivity of 91.15%to discriminate HCC from CHB groups than serum alpha-fetoprotein(AFP)alone(91.15%vs65.49%,P=0.000).Then we compared the diagnostic value of the combination of DKK3 and ITIH5 methylation with AFP alone in detecting early HCC.We found the sensitivity of methylated ITIH5 and DKK3 combination showed 86.49%,higher than serum AFP levels(86.49%vs54.05%,P=0.002)in detecting early HCC.ConclusionsOur findings strongly showed that the methylation of DKK3 and ITIH5 in PBMCs may involved in the progression of HCC.DKK3 combined ITIH5 methylation in PBMCs greatly improved the sensitivity of detection of HCC and may serve as a useful noninvasive biomarker for detecting early HCC and HCC metastasis.