Methylation Of Protocadherin10in Hepatocellular Carcinoma And Its Relationship With Hepatitis B Virus

Hepatocellular carcinoma(HCC) is characterized by highly malignantwith rapid progression[1], the mortality rate of HCC ranks the third and forthplace respectively in male and female cancer deaths[2].Thus, theprevention,early diagnosis and novel therapeutic strategies for HCCbecome the urgent need.Hepatocarcinogenesis is a mutual-synergized consequence ofaccumulative genetic and epigenetic abnormalities in the genome of livercells[3-6].As a major risk factor in the development of HCC[7,8],chronic andpersistent infection of hepatitis B virus (HBV) is frequently associated withvarious epigenetic changes,among which inhibition of some tumorsuppressor genes by oncogenic X protein of HBV (HBx) via inducinghypermethylation of CpG islands in the promoter region could be animportant step in carcinogenesis[9,10].Therefore,evaluation of HBV/HBxmediated DNA methylation based molecular biomarkers have potentialclinical applications in early detection, diagnosis, and targeted therapies ofHBV-related HCC. Protocadherin10(PCDH10), also known as OL-protocadherin, is anewly identified protocadherin of the cadherin family which function asimportant factor through maintenance of cell/cell adhesion[11,12].A recentstudy demonstrated a tumor suppressor role of PCDH10with inhibition ofthe growth,migration,and invasion of several human cancers cells uponoverexpression[13].The gene locus of PCDH10at4q28.3is frequently deletedin hepatoma,colorectal cancer, prostate cancer, pancreaticcancer[14-17].Recently, the hypermethylation of CpG islands in the PCDH10gene promoter was found in hepatoma[13].However,the role of infection ofhepatitis B virus or HBx in this process remains unclear at thistime.Therefore,our study aims to further investigation of the potentialrelationship between HBV/HBx and hypermethylation of PCDH10promtertogether with the downregulation of PCDH10transcriptional level bystudying several human hepatocellular carcinoma cells, HepG2cells withHBx transfectants and HCC tissues through reverse-transcriptionpolymerase chain reaction(RT-PCR),methylation-specific polymerase chainreaction(MSP) and bisulphite genomic sequencing(BGS).Our study demonstrated PCDH10expression was found to berepressed or down-regulated in9/13(69.2%) of the HCC cell linescompared with normal adult liver tissue.Among which the transcriptionalexpression of PCDH10was silenced in hepatocellular carcinomacells:HepG2、Huh4、Huh7and Snu449,while down-regulated defferently in Huh1、Huh6、Mahlavu、PLC/PRF/5and Snu397.Treatment with the DNAmethyltransferase inhibitor5-aza-2’-deoxycytidine (Aza) was sufficient torestore PCDH10mRNA expression by partly suppressing PCDH10promoter methylation in HepG2cells.PCDH10methylation was furtherdetected in76%(38of50) of HCC tissues compared with40%(20of50)of paired nontumor tissues (P <0.01), with no methylation detected innormal human liver tissues.Correspondingly, decreased PCDH10expression was further detected in64%of HCC tissues,30%of pairednontumor tissues (p＜0.05)and no normal human liver tissues.All thedetails revealed that decreased PCDH10expression was correlated withhypermethylation of the PCDH10promoter.Through the further investigation about PCDH10mRNA expressionand the methylation status of PCDH10gene in our panel of23HCC tissueswith hepatitis B and27HCC tissues without HBV infection,we found thatdecreased PCDH10mRNA expression is relevant to infection of HBV(p＜0.01),however, there was no correlation between the methylation status ofPCDH10promoter and chronic HBV(p＞0.05).Meanwhile, the PCDH10promoter methylation status was found not to be associated with HBxexpression in HepG2-HBx transfectants.Though there was no correlation between the methylation status ofPCDH10and clinicopathological features such as age, sex, chronic HBVinfection, ethanol abuse and cirrhosis, well correlations between the methylation status of PCDH10and tumor size, serum AFP levels,metastasis(p＜0.05) and TNM staging(p＜0.01) indeed existed.Thus,PCDH10may function as a hypermethylation biomarker of potentialprognostic in HCC, being a good indicator of clinical outcome.