Epigenome-wide DNA Methylation Profiling Of Portal Vein Tumor Thrombosis (PVTT) And The Methylation Patterns Of DNAH17 And ADRA1A In Hepatocellular Carcinoma

Background Hepatocellular carcinoma(HCC)is one of the most aggressive human malignancies and one of the leading causes of cancer death in China.The early diagnosis and treatment of HCC remain big challenges to us.Therefore,it is crucial to find efficient biomarkers and new mechanism of HCC initiation and progress.Portal vein tumor thrombosis(PVTT)is a special pattern of metastasis and hard to deal with during the clinical practice.Aberrant methylation has recently received increasing attention because these alterations are relatively stable but potentially reversible therapeutically.However,the epigenome-wide methylation patterns of PVTT have not been fully explored.Here,our study analyzed the DNA methylation profiling of portal vein tumor thrombosis(PVTT)tissues in hepatocellular carcinoma patients.We also analyzed the methylation patterns of DNAH17 and ADRA1 A in HCC.Method We performed epigenome-wide DNA methylation profiling of adjacent normal tissues(ANTs),HCC tissues and PVTTs using an Infinium Human Methylation450 array(patient number = 11).We also conducted MTS assay and apoptosis assay to assess the synergistic effect of decitabine(DAC,DNA methyltransferase inhibitor)and tumor necrosis factor-related apoptosis-inducing ligand(rh-TRAIL)on the HCC cell lines.We evaluated the methylation levels of DNAH17 in 163 HCC samples and their paired normal tissue using Sequenom Epi TYPER assays and performed the Taq Man copy number assay to assess the copy number status of DNAH17 in HCC samples.The methylation levels of ADRA1 A in HCC samples and ANT were also detected by Sequenom Epi TYPER assays.Immunohistochemistry,q PCR and RNA-seq were used to assess the expression levels of DNAH17 and ADRA1 A.Results 1.The mean global methylation levels of HCC tissues and PVTTs were significantly lower than ANTs(P<0.01).A total of 864 differentially methylated Cp G sites annotated in 532 genes were identified between HCC tissues and paired PVTTs(|mean methylation difference| >10%,P<0.005).The pathway analysis based on hypermethylated genes in PVTT tissues was interestingly significantly enriched in regulation of actin cytoskeleton pathway(P=4.48E-5).We found 23 genes whose methylation levels were gradually alternated in HCC and PVTT,with 17 genes being increased(eg.PAK1,NDRG2,SLC9A3R2,ZC3H3)and 6 genes decreased(ADORA2A,KIAA1143,NAPEPLD,PARVG,TNFRSF10 A,TSTD1).TNFRSF10 A is one of the key cell surface receptors of TRAIL and could be activated by rh-TRAIL.The MTS assay and apoptosis assay demonstrated the combination of DAC and rh-TRAIL could synergistically suppress the proliferation and induced apoptosis in SK-Hep-1 and Huh7 cell lines.2.The mean methylation levels were significantly decreased in the tumor tissues compared to the paired normal tissues in both selected regions of DNAH17(amplicon 1: 58.7% vs.84.5%,P<0.0001;amplicon 2: 69.9% vs.84.5%,P=0.0060).Contrarily,both RNA-seq and immunohistochemistry indicated the expression of DNAH17 were increased in tumor tissues(P<0.05).DNMT inhibitor decitabine treatment could increase the expression of DNAH17 in HCC cell lines.DNAH17 gene amplification always companied with hypomethylation status.Moreover,hypomethylation status was associated with several clinical characteristics,such as male patients,higher AFP values,higher age of onset,fibrous capsules,tumor necrosis,liver cirrhosis and tumor thrombus(P<0.05).Receiver operator characteristic(ROC)curve analysis demonstrated the methylation levels of DNAH17 could efficiently predict the existence of the fibrous capsule(AUC=0.695)and tumor thrombus(AUC=0.806).3.Compared with that in paired normal tissues,the mean methylation level of the ADRA1 A promoter region was significantly increased in tumor tissues from 160 HCC patients(25.2% vs.17.0%,P<0.0001).We found that a DNA methyltransferase inhibitor(decitabine)could increase the expression of ADRA1 A m RNA in HCC cell lines.Moreover,hypermethylation of the ADRA1 A gene in HCC samples was associated with clinical characteristics,including alcohol intake(P=0.0097)and alpha-fetoprotein(P=0.0411).Receiver operator characteristic(ROC)curve analysis demonstrated that the mean methylation levels of ADRA1 A could discriminate between HCC tissues and adjacent non-cancerous tissues(AUC=0.700,P<0.0001).m RNA sequencing indicated that the main enriched pathways were pathways in cancer,cytokine-cytokine receptor interaction and metabolic pathways(P<0.01).Conclusion Our findings indicated that DNA methylation plays an important role in the PVTT formation through regulating the metastasis-related pathways.The combination of DAC and rh-TRAIL could be a promising treatment strategy for HCC.Aberrant methylation of DNAH17 was associated with comprehensive HCC clinicopathological factors and could be a promising biomarker for tumor thrombosis in HCC patients.ADRA1 A gene hypermethylation might contribute to HCC initiation and is a promising biomarker for the diagnosis of HCC.