Detecting abnormal methylation of tumor suppressor genes GSTP 1 , P 16 , RIZ 1 , and RASSF 1 A in hepatocellular carcinoma and its clinical significance

Hepatocellular carcinoma (HCC) has a high rate of mortality. Further studies into epigenetic changes in HCC, particularly the abnormal methylation of tumor suppressor genes (TSGs), are required, since these changes may provide novel biomarkers for early screening and diagnosis of HCC. By using methylation-specific polymerase chain reaction (MSP), the present study detected the methylation status in the promoter region of 4 candidate TSGs, GSTP1, P16, RIZ1, and RASSF1A, respectively, in 35 paired HCC and tumor-adjacent liver tissues in addition to 20 normal liver tissues. Their effect on the initiation and progression of HCC was also investigated by analyzing the clinicopathological data. The results of the present study revealed that the methylation level of RIZ1 and GSTP1 genes in HCC was significantly increased compared with that in the adjacent tissues (P<0.01) and the normal liver tissues (P<0.01). The methylation frequency of P16 and RASSF1A genes was not significantly increased compared with that observed in the adjacent tissues (P>0.05) but was significantly increased compared with the normal tissues (P<0.01). In HCC tissues, the methylation frequency of the GSTP1 gene in tumors with capsular invasion was significantly increased compared with that in tumors without capsular invasion (P<0.05). The methylation frequency of P16 gene in hepatitis B surface antigen (HbsAg)-positive HCC patients was significantly increased compared with that in HbsAg-negative patients (P<0.05). The methylation status of RIZ1 and RASSF1A genes was not significantly correlated with the clinicopathological data (P>0.05). Previous studies have demonstrated that the methylation status of RIZ1 and GSTP1 genes is HCC‐specific, and thus may be used as a biomarker to assist the clinical diagnosis of HCC. While the methylation of GSTP1 gene promoter may associate with the invasiveness of HCC, chronic hepatitis B virus infection may be the cause of methylation-induced P16 inactivation.


Introduction
HCC is one of the tumors with the highest incidence worldwide, and its incidence and mortality in China have remained high.The occurrence of HCC is results from multiple factors, including the activation of certain oncogenes, inactivation of TSGs and exogenous stimuli.Previous studies have demonstrated that the hypermethylation of CpG islands in tumor suppressor gene (TSG) promoters is closely associated with the formation of HCC, which may transform the spatial structure of chromatin and hence block/silence the transcription of TSGs via recruiting proteins of the methyl CpG binding domain (MBD) family in addition to the associated protein complexes (1,2).Epigenetic silencing is a relatively common mechanism of TSG inactivation (3).The aberrant methylation of tumor-associated genes, particularly TSGs, requires further research.
A high frequency of methylation inactivation of the GSTP1 gene has been observed in human prostate, kidney, and liver cancers (4)(5)(6).Zhang et al (7) and Tchou et al (8) demonstrated that there is a high frequency of methylation events in the GSTP1 gene in HCC tumor samples and HCC cell lines, and that the methylation of GSTP1 in HCC is associated with the action of environmental carcinogens.As a TSG, P16 gene inactivation may result in excessive cell proliferation, and the promoter methylation on 9p21 in HCC patients represents the most common mechanism of P16 inactivation (9).Zhong et al (10) revealed that the abnormal methylation of the RASSF1A gene promoter is present in 95% of HCC tissues; the authors hypothesized that the change in RASSF1A gene expression is an early event during hepatitis B virus-induced tumorigenesis of HCC.
The present study comparatively analyzed the changes in methylation level of 4 TSGs in samples from HCC tumors, tumor-adjacent tissues, and normal liver tissues, and

Materials and methods
Written informed consent was obtained from the families of all patients, and the Human Research Ethics Committee of the Affiliated Nanjing University Drum Tower Hospital (Nanjing, China) approved the use of all samples under a protocol that conforms to the provisions of the Declaration of Helsinki (as revised in Seoul, 2008).
Specimens.The tumor specimens were collected from HCC patients who had undergone surgical treatment in the Department of Hepatobiliary Surgery of the Affiliated Drum Tower Hospital, School of Medicine, Nanjing University, in the Changzhou First People's Hospital, and in the Third Affiliated Hospital of Soochow University, during the period between January 2013 and January 2014.The patients did not receive any anticancer treatment and had no other endocrine, immune, and metabolic diseases prior to the surgery.Any hemorrhagic and necrotic regions were avoided during the tumor sample collection.The tumor-adjacent liver tissues were obtained from the area within 1.5 cm distance from the edge of HCC, and histologically confirmed for no infiltration of cancer cells.The normal control group contained 20 cases of pathologically confirmed normal liver tissue.All the specimens were frozen in liquid N 2 immediately following the resection, then transported, and stored at -80˚C.
Methylation-specific polymerase chain reaction (MSP) and result interpretation.The DNA samples were extracted from the liver specimens using a DNA extraction kit (Sangon Biotech Shanghai Co., Ltd., Shanghai, China), following the manufacturer's instructions.The concentration and purity of the extracted DNA were measured on a UV spectrophotometer (UV-240; Shimadzu Corp., Kyoto, Japan), and suitable DNA samples were stored at -80˚C.
Bisulfite modification of the DNA samples was conducted using anEZ DNA Methylation-Direct TM Kit (Zymo Research, Irvine, CA, USA) according to the kit instructions.The DNA samples were then amplified by MSP and analyzed for methylation status based on the differential amplification pattern.
The primer sequences, annealing temperature, and product sizes are presented in Table I.The PCR system contained PCR Mixture 2X Mix 15 µl, U or M-Primer F 0.5 µl, U or M-Primer R 0.5 µl, Modified DNA 1-5 µl; the total volume was adjusted to 30 µl with deionized H 2 O.The PCR conditions were as follows: 94˚C denaturation, 3 min; 94˚C denaturation 30 sec, annealing 30 sec, and 72˚C extension 30 sec, 40 cycles; 72˚C extension 7 min.The amplification product was stored at 4˚C.The PCR products (10 µl each) were separated by 2% agarose gel electrophoresis, and the images were captured on a gel imaging and analysis system (Yu Long Co., Ltd., Kunming, China).
Samples that amplified from the primer pair for methylated DNA sequence were considered methylation-positive, whereas samples that amplified from the primer pair for unmethylated sequence were considered methylation-negative.Samples with PCR products from both primer pairs were considered partially methylated, which also represents a methylation-positive status.
Statistical analysis.The data were statistically analyzed using SPSS statistical software, version 17.0 (SPSS, Inc., Chicago, IL, USA).The count and measurement data were compared by χ 2 test and t-test respectively.The association between the gene methylation in HCC tissues and the clinical data was analyzed using a Fisher's exact probability test.P<0.05 was used to indicate a statistically significant difference.
The frequency of GSTP1 gene methylation in HCC with capsular invasion was significantly increased compared with in HCC without capsular invasion (P<0.05).Among the HCC samples, the frequency of P16 gene methylation in HbsAg-positive HCC tissues was significantly increased compared with those in HbsAg-negative HCC tissues (P<0.05).The methylation status of RIZ1 and RASSF1A genes did not demonstrate significant correlation with the clinicopathological data of patients (P>0.05;Tables III and IV).

Discussion
HCC is one of the tumors with highest incidence worldwide.Due to an insidious onset, the majority of HCC patients are diagnosed at an advanced stage, which results in a <20% clinically resectable rate (11,12).With the exception of α-fetoprotein (AFP), at present, there remains a lack of well-recognized effective tumor markers for routine clinical detection of HCC.Therefore, identifying HCC-associated tumor markers and studying the underlying molecular mechanisms is of great importance.Epigenetics refers to a genetic mechanism that enables the occurrence of inheritable alterations in gene expression without changing the DNA sequence (3).Epigenetic Table I.Primers for methylation-specific polymerase chain reaction.

Methylation status
Table III.The correlation between the methylation state of GSTP1 and P16 genes with the clinicopathological data of HCC patients.
RIZ1 is a TSG that simultaneously regulates cell proliferation and inhibits oncogenesis (15).The abnormal expression of RIZ1 is associated with the tumorigenesis of a variety of human tumors, including nasopharyngeal, breast, liver and cervical among other cancers (16,17).Nomoto et al (18) reported that the frequency of RIZ1 gene methylation was 45.2% among patients with HCC in Japan.The results of the present study demonstrated that the frequency of RIZ1 methylation in HCC (68.6% [24/35]) was significantly increased (P<0.01)compared with in the paired adjacent liver tissues (14.3% [5/35]), while no methylation of RIZ1 was detected in the normal liver tissues.The frequency of RIZ1 methylation demonstrated a significant gradient among the 3 groups of samples; the rare presence of RIZ1 in the adjacent Table IV.The correlation of the methylation state of RIZ1 and RASSF1A genes with the clinicopathological data of HCC patients.
In summary, by analysis of the methylation frequency of 4 TSGs in HCC, tumor-adjacent, and normal liver tissues, the present study revealed a progressive epigenetic change during the formation of HCC, which reflected the molecular mechanism of the multi-step and multi-stage origin of HCC.In addition, the results demonstrated that the status of RIZ1 and GSTP1 gene methylation has good specificity for HCC, may better distinguish between HCC and non-cancerous tissues, and may therefore be used as novel candidate biomarkers to assist the early screening and diagnosis of HCC.With further study on TSG methylation and larger sample sizes in the future, a more thorough insight may be gained into the effect of abnormal methylation of TSGs on the occurrence and development of HCC.

Figure 2 .
Figure 2. The MSP products of GSTP1, P16, RIZ1, and RASSF1A genes in part of the HCC, paired tumor-adjacent, and normal liver tissues.The lowest marker band represents 100 bp.N, PCR negative control; M, methylated; and U, unmethylated.a, cancerous tissue; p, adjacent liver tissue; and n, normal liver tissue.
Damman et al in 2000ghly tumor-specific epigenetic changes in RIZ1 with the occurrence of HCC, indicating that the methylation of RIZ1 may be a frequent event during the tumorigenesis of HCC, and that the appearance of RIZ1 methylation may indicate an immediate formation of HCC.Therefore, RIZ1 gene methylation may be of great significance for the risk assessment and early diagnosis of HCC.However, further analysis indicated that there was no correlation between RIZ1 methylation and the clinicopathological data of HCC patients.It should be noted that since the exact role of RIZ1 promoter methylation in the process HCC carcinogenesis remains unclear, further study with an expanded sample size is required.RASSF1A is a TSG cloned from lung cancer and reported byDamman et al in 2000 liver