Predictive Value Of DNA Methylation Of Tumor Suppressor Genes In Tumor Recurrence Of Patients With Hepatocellular Carcinoma After Liver Transplantation

Hepatocellular carcinoma (HCC) is the most common hepatic malignant tumor, ranking the fifth among tumors in the world. Our country is a big country for hepatitis B, with a high carrying rate of hepatitis B virus in the population. As the terminal end of the progression chain of hepatitis-liver cirrhosis-HCC, the high incidence of HCC is an indisputable fact, making our country the place with the highest HCC incidence in the world. Undeniably, after persistent effort in the past decades, our country has obtained remarkable achievements in the diagnosis and treatment of HCC. However, various comprehensive treatments on the basis of surgicial excision (including radiotherapy, chemotherapy, biological, targeted treatment and liver transplantation) still can not resolve the general poor prognosis of HCC. With development of hepatological surgery and standardization of inclusion criteria for HCC patients, liver transplantation is considered an ideal therapy for HCC. However, tumor metastasis and recurrence after transplantation still greatly restrict the therapeutic effect. In the recent years, arrounding recurrence and metastasis after liver transplantation, studies on biomarkers of HCC recurrence have become an important research aspect.Meanwhile, with deeper understanding of tumor metastasis and development of human genomics, it is realized that the pathogenesis and progression of HCC is a complex process involving changes and regulation of multiple genes. Studies related to malignant tumor gene change and regulation have become a hot pot. As the most important content of the newly emerged epigenetics, inactivated transcription of tumor suppressor gene caused by DNA methylation, which further impacts tumor development has attracted many researchers'attentions. However, through literature review, we find that there is no report of predictive value of the new tumor suppressor gene PCDH10 and CIMP in tumor recurrence of patients with hepatocellular carcinoma after liver transplantation. Thus, on the basis of previous research, we first attempted to establish CIMP to reflect the biological features of HCC and discuss its predictive value in tumor recurrence after liver transplantation; we also used the novol TSG- PCDH10 to systemically investigate the role and regulation mechanism of candidate TSGs for optimized CIMP in the development of HCC, so as to establish a CIMP model closedly related to recurrence, further improve early prediction of recurrence after liver transplantation, establish HCC liver transplantation "China standard" and provide new candidate tumor biological parameters.Part I Predictive value of DNA methylation of tumor suppressor genes and related CpG island methylator phenotype in tumor recurrence of patients with hepatocellular carcinoma after liver transplantationObjective:The promoter methylation of gene is an important epigenetic modification, which can bind methyl-CpG-binding domain (MBD) and histone acetylation cause gene expression silence, especially the methylation of tumor suppressor gene causing expression silence which plays an extremely important role in carcinogenesis and progression. CpG island methylator phenotype (CIMP), in which multiple genes are concurrently methylated has been recognized as one of the important mechanisms in hepatocellular carcinoma and associated with elevated serum alpha-fetoprotein level. We wanted to investigate the promoter methylation of gene, confirm CIMP associated prognositic in HCC and analyze relationship between CIMP status and clinicopathologic features.Methods:Methylation status of CpG islands on 12 tumor suppressor gene promoter regions were studied, including P16, CDH1, GSTP1, DAPK, MGMT, XAF1, TIMP3, SOCS1, SFRP1, TMS1, SYK and DKK1 (these genes are known to be associated with different human malignant tumors, especially carcinogenesis, invasion, metastasis and even prognosis of gastrointestinal tumor). Preliminary screening with Methylation-Specific PCR (MSP) was first performed to assess the DNA methylation status of tumor tissues from 20 patients with HCC treated with liver transplantation.7 tumor suppressor genes (P16, CDH1, GSTP1, DAPK, XAF1, SOCS1, SYK) whose methylation frequency was higher than 40% were selected as the marker genes of CIMP. Methylation detection of the above sites was expanded to tumor tissues from 65 patients with HCC treated with liver transplantation. Then according to the methylation status, CIMP classification was conducted for these 65 patients. On the other hand, clinical pathological materials of the patients with HCC liver transplantation were collected. Follow up was performed to evaluate the relationship between CIMP and clinical pathological parameters and 3-year recurrence-free survival after transplantation.Results:The methylation frequencies of 12 gene in our clinical samples were different from 5% for MGMT and 66% for XAF1; among the 7 selected marker genes of CIMP, there was concordant methylation phenomenon, i.e. P16 and SYK were hypermethylated concurrently (P=0.001), P16 and CDH11 were hypermethylated concurrently (P=0.008), CDH1 and DAPK were hypermethylated concurrently (p=0.003). After analyzing individual tumor suppressor gene, clinical pathological parameters and prognostic information, it was found that hypermethylation of GSTP1 was associated with low histologic grade (P=0.003) and multiple tumor numbers (P=0.011); hypermethylation of XAF1 was associated with pre-operative AFP level (P=0.007); besides, methylation status of DAPK was associated with tumor size (P=0.016). There was no significant relationship between methylation status of other individual genes and clinical pathological parameters. No statistic relationship was found between the methylation status of individual genes and tumor recurrence (P>0.05). Log-rank test was used to analyze relationship between number of hypermethylated genes and recurrence. The results indicated that among the 7 selected tumor suppressor genes, there was the most significant relationship between hypermethylation of≥3 genes and HCC recurrence after liver transplantation (P=0.004). Therefore, CIMP was classified into CIMP positive (hypermehtylation of≥3 genes) and CIMP negative (hypermethylation of< 3 genes). The subsequent analysis showed that there was correlation between CIMP status and pre-operative AFP level as well as tumor size (P=0.017 and 0.007 respectively). However, CIMP status was not related to other clinical pathological parameters, such as gender, age, PVTT, histological grade or tumor size (P>0.05). Meanwhile, in combination with Liver Transplantation Hangzhou Criteria proposed previously by our center, it was confirmed that patients beyond Hangzhou Criteria had the higher CIMP positive rate than patients meeting Hangzhou Criteria (84% vs.57%, P=0.017). Then, we used Kaplan-Meier survival curve to analyze the relationship between CIMP and survival of patients with HCC liver transplantation. It was found that 3-year currence-free survival after liver transplantation of patients in CIMP+group was shorter than that of patients in CIMP-group, with significant difference (15.3 months vs.30.3 months, P=0.004). Results of Cox univariate analysis showed that besides pre-operative AFP level and tumor size, CIMP status was also related to recurrence. Multivariate analysis indicated that besides tumor size, CIMP status (risk coefficient:3.581; 95%CI:(1.473-8.710); P=0.005) was also an independent risk factor influencing recurrence.Conclusion:CIMP was an independent risk factor influencing HCC recurrence after liver transplantation and thus had potential value in predicting HCC recurrence after liver transplantation.Part II The study of a novel tumor suppressor gene, PCDH10 in hepatocellular carcinoma.Objective:Protocadherin 10 (PCDH10) is a newly identified protocadherin of the cadherin family. Early studies on PCDH 10 mainly focused on neurophysiology. It is considered that PCDH10 is essential for brain development, especially in elongating striatal axons and guiding thalamocortical projections. Recent studies indicated that in nasopharyngeal carcinoma, leukemia, cervical carcinoma, breast cancer and gastric carcinoma cell strains and tissues, there was expression loss caused by hypermethylation of PCDH 10 promoter region. Through exogenous over expression of PCDH 10 protein, the proliferation, invasion and metastasis of tumor cells could be significantly inhibited and apoptosis of tumor cells could be induced. In the study of cervical carcinoma, it was considered that hypermethylation of PCDH10 could serve as a potential specific diagnostic marker of cervical carcinoma. In the latest clinical study on gastric carcinoma, it was not only confirmed that PCDH10 was a typical tumor suppressor gene but also that hypermethylation of PCDH10 was closely associated with poor prognosis. However, there is no report on clinical and basic research of PCDH10 in hepatocellular carcinoma and the biological effect of PCDH10 protein in hepatocellular carcinoma cells is still unknown.Methods:Western blotting and immunohistochemistry were used to detect expression of PCDH10 in tumor tissues and paraneoplastic tissues from HCC patients as well as normal liver tissues. RT-PCR was used to detect PCDH10 mRNA expression level in hepatocellular carcinoma cells before and after application of demethylation agent 5'-Aza-dC. The mechanism of epigenetic regulation was studied. Meanwhile, MSP and BSP were used to verify methylation status change of the target genes. The correlation between PCDH10 methylation and clinical pathological parameters as well as recurrence was analyzed in 65 patients with HCC liver transplantation; RNA interference and plasmid transfection were used to respectively mimic PCDH10 absence and over expression, so as to study the effect of PCDH10 protein on proliferation, apoptosis, invasion and clone formation ability of hepatocellular carcinoma cells in both sides.Results:Among our selected 5 hepatocellular carcinoma cell lines,80% cell lines had reduced or absent PCDH10 mRNA expression. Hypermethylation of PCDH10 promoter region was the main reason resulting in gene expression loss. In clinical specimens, it was found that PCDH10 expression loss caused by DNA methylation was tumor specific, i.e. the expression in hepatocellular carcinoma tissues was relatively lower than that in paraneoplastic tissues and normal tissues. It was also revealed that patients with HCC liver transplantation with hymethylation of PCDH10 were more common to have recurrence after operation and had poor prognosis. In vitro experiment also found that over expression of PCDH10 could inhibit proliferation of hepatocellular carcinoma cell lines, reduce cellular invasion and clone formation, and induce apoptosis.Conclusion:In hepatocellular carcinoma, DNA methylation of PCDH10 promoter region is a main factor causing loss of protein expression but not the sole factor; methylation status of PCDH10 is a potential biological marker for HCC recurrence after liver transplantation. Over expression of PCDH10 protein can significantly inhibit growth of tumor cells, promote apoptosis of HCC cells and reduce cell invasion and clone formation.