The Mechanism Of Aberrant Oct-6, Sall3 CpG Island Methylation Regulating MRNA Expression In Human Hepatocellular Carcinoma

Background and ObjectiveHuman hepatocellular carcinoma (HCC) is one of the most common gastrointestinal malignancies, ranking the fifth in occurrence of common cancer, the third in common cause of cancer-related death. HCC has become a major damage to human health. The prognosis of HCC is very poor and the reported overall five-year survival after resection is only 30%-60%. That may be caused by difficult diagnosis of early HCC. The diagnosis of HCC is usually made by serum AFP. But AFP is elevated in not all the patients with HCC. The AFP level in 30%-40% of patients (especially small HCC) is negative or less and AFP is also elevated in patients with chronic hepatitis or cirrhosis. AFP as a screening marker can result in misdiagnosis, so new and more specific diagnosis methods are urgently needed.DNA methylation is a regulatory mechanism of gene expression and a contributor to carcinogenetic process. Aberrant CpG island methylation of tumor-related gene is an early and frequent event in carcinogenetic process and plays an important role in the development of cancer. Researches on CpG island methylation of specific genes may be a new perspective to clarify the molecular mechanism of HCC. To make individualized treatment and improve efficacy, we should focus on looking for HCC methylation profiling which may be used for early diagnosis and MDR (multi-drug resistance, MDR) screening.Some papers reported that aberrant methylation of Oct6 is associated with tumor differentiations of NSCLC (Non-small cell lung Cancer) and methylation profiling of sall3 may be provides a sensitive and specific detection for bladder cancer. It also has been reported that Oct6 gene and sall3 gene CpG island have higher frequency of hypermethylation in HCC tumor tissues compared to adjacent non-cancerous tissues by methylation qualitative method. So aberrant methylation of Oct6 and sall3 CpG island may be associated with HCC carcinogenesis. As two new targets of methylation profiling, their quantitative methyltion levels, mRNA expression levels, transcription regulatory mechanism and clinical significance of aberrant methylation in HCC remains unkown.In this study, we quantify the CpG island methylation levels of Oct6 and sall3 in 38 tumor tissues and adjacent non-cancerous tissues by MassArray platform; we also detect Oct6 and sall3 mRNA levels in the 38 paired samples by Real-time PCR to investigate the relationship between CpG island methylation and mRNA expression. To further confirm Oct6 and sall3 CpG island methylation regulate mRNA expression, we also analyzed the changes of CpG island methylation levels and mRNA levels after O.1,0.5,2.5μM 5-Aza-2-deoxycytidine (5-Aza-2'-deoxycytidine,5-Aza-CdR) treatment in Huh7, HepG2, SMMC7721, Be17402, SK-HEP1 and QGY7703 cell lines. The methylation levels of Oct6 and sall3 in different groups of age, gender, tumor differentiations and hepatitis virus infection were also analyzed and provides preliminary theory basis for clinical significance of Oct6 and sall3 aberrant methylation.Materials and Methods1. SubjectsThirty-eight paired clinical samples of HCC-including tumor tissues and adjacent non-cancerous tissues were collected from surgical specimens at Department of Hepatobiliary Surgery of Nan fang Hospital (16 cases) and Cancer Institute of Sun Yat-sen University (22 cases). Six HCC cell lines (Huh7, HepG2, SMMC7721, Be17402, SK-HEP1 and QGY7703) were obtained from Shanghai Institute of Cell Biology. All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum and incubated under a 5% CO2 atmosphere at 37℃.2. Cell lines were treated by 5-Aza-CdRExponential growth phase of hepatoma cell lines were seeded into 6-well plates. After 24 h of culture, cell lines were treated with 5-Aza-CdR at concentrations of 0, 0.1,0.5 and 2.5μM for 3 days and culture medium was replaced every 24 h with fresh media containing 5-Aza-CdR.3. Quantitative DNA methylation detectionGenomic DNA was extracted from HCC cell lines and clinical samples using DNA extraction kit. Genomic DNA was modified with sodium bisulfite. Quantitative methylation analysis of 38 clinical samples-including tumor tissues and adjacent non-cancerous tissues and six cell lines treated by 5-Aza-CdR or not were determined by the MassArray(?)EpiTYPER platform (Sequenom).4. Quantitative mRNA detectionWe used Real-time PCR to evaluate the mRNA expression levels of Oct6 and sall3 in 38 clinical samples-including tumor tissues and adjacent non-cancerous and six cell lines treated by 5-Aza-CdR or not. Relative levels of Oct6 mRNA were calculated using 2△△Ct.β-actin was used as an internal control. Data were expressed from three separate experiments.5. Correlation analysis between methylation levels and transcription levelsThe relationship between methylation levels and transcription levels was analyzed using the Spearman test. All tests were two-sided; significant level was assigned atα=0.05. All the date was analyzed by SPSS 13.0. 6. Methylation levels analysis in different groupsThe methylation levels of Oct6 and sall3 in tumor tissues and adjacent non-cancerous tissues were compared by Wilcox rank sum test. The methylation levels in different groups of age, gender, hepatitis virus infection were compared by Wilcox rank sum test. The methylation levels in three different tumor differentiations were compared by Kruskal-Wallis test. All tests were two-sided; significant level was assigned at a=0.05. All the date was analyzed by SPSS 13.0.Results1.1 CpG island methylation levels and mRNA levels of Oct6 in HCC tumor tissues and adjacent non-cancerous tissues1.1.1 CpG island methylation of Oct6 in HCC tumor tissues and adjacent non-cancerous tissues47.3% (18/38) of the tumor tissues exhibited higher methylation levels of Oct6 CpG island compared to adjacent non-cancerous tissues. The average methylation levels of each CG unit increased from 14% to 26% in tumor tissues as compared to adjacent non-cancerous tissues.52.7% (20/38) of patients exhibited similar methylation levels in tumor tissues and adjacent non-cancerous tissues.1.1.2 Oct6 mRNA levels in HCC tumor tissues and adjacent non-cancerous tissues81.6% (31/38) of the tumor tissues showed lower Oct6 mRNA levels than adjacent non-cancerous tissues.3.2% (5/38) of the tumor tissues showed similar mRNA levels with adjacent non-cancerous tissues and 5.2% (2/38) of the tumor tissues exhibited higher mRNA levels than adjacent non-cancerous tissues.1.1.3 The relationship between the CpG island methylation and mRNA expression of Oct6 in tumor tissues and adjacent non-cancerous tissuesAmong the 31 tumor tissues with lower Oct6 mRNA levels than adjacent non-cancerous tissues,17 exhibited higher methylation levels and a negative association was found between the CpG island methylation and mRNA expression (rs=-0.789, P<0.001); 14 tumor tissues had similar methylation levels with adjacent non-cancerous tissues. One tumor tissue with higher mRNA level had similar methylation level with the adjacent non-cancerous tissue. One tumor tissue with higher mRNA level exhibited higher methylation level than adjacent non-cancerous tissue. Five tumor tissues had no significant difference in mRNA levels and methylation levels compared with adjacent non-cancerous tissues.1.2 5-Aza-CdR induces the changes of Oct6 CpG island methylation levels and mRNA levels in HCC cell linesTo further identify the DNA methylation is one of Oct6 transcription regulatory mechanisms, we also analyzed whether demethylating agent up-regulated Oct6 mRNA levels through down-regulating CpG island methylation levels. After treatment with 0.1,0.5,2.5μM 5-Aza-CdR, Oct6 CpG island is dose-dependently down-regulated and Oct6 mRNA levels is dose-dependently up-regulated in Huh7 and Be17402 cell lines; Oct6 CpG island is dose-dependently down-regulated but Oct6 mRNA levels is irregularly up-regulated in SK-HEP1 and QGY7703 cell lines; Oct6 CpG island methylation were dose-dependently down-regulated in SMMC7721 cell line, but there were no significant alterations in Oct6 mRNA levels. In HepG2 cell line, the methylation levels of some CG units were less than 20%; after 5-Aza-CdR treatment, Oct6 CpG island methylation levels slightly decreased and mRNA levels slightly increased.1.3 The methylatioin levels of Oct6 CpG island in different clinicopathological feature in HCCThe methylation levels of Oct6 CpG island were higher in female than in male and 5 CG units were significantly different;>48 years group exhibit higher methylation levels than≤48 years group and 6 CG units were significantly different; HBV negative group had higher methylation levels than HBV positive group and 6 CG units were significantly different. There was no significant correlation between Oct6 CpG island methylation levels and tumor differentiation. 2.1 CpG island methylation levels and mRNA levels of sall3 in HCC tumor tissues and adjacent non-cancerous tissues2.1.1 CpG island methylation levels of sall3 in HCC tumor tissues and adjacent non-cancerous tissues71%(27/38) of the tumor tissues exhibited higher methylation levels of sall3 CpG island than adjacent non-cancerous tissues. The average methylation levels of each CG unit increased from 15% to 37% in tumor tissues compared to adjacent non-cancerous tissues.29%(11/38) of tumor tissues exhibited similar methylation levels with adjacent non-cancerous tissues.2.1.2 sall3 mRNA levels in HCC tumor tissues and adjacent non-cancerous tissues86.8% (33/38) of the tumor tissues showed that lower sall3 mRNA levels than adjacent non-cancerous tissues.5.3% (2/38) of the tumor tissues showed similar sall3 mRNA levels with adjacent non-cancerous tissues and 7.9% (3/38) of the tumor tissues exhibited higher sall3 mRNA levels than adjacent non-cancerous tissues.2.1.3 The relationship between the CpG island methylation and mRNA expression of sall3 in tumor tissues and adjacent non-cancerous tissuesAmong the 33 tumor tissues with lower mRNA levels than adjacent non-cancerous tissues,24 exhibited higher methylation levels and a negative association was found between the CpG island methylation and mRNA expression (rs=-0.714, P<0.001); 9 tumor tissues had similar methylation levels with adjacent non-cancerous tissues. One tumor tissue with higher mRNA level had similar methylation level with the adjacent non-cancerous tissue. Two tumor tissues with higher mRNA levels exhibited higher methylation levels than adjacent non-cancerous tissue. One tumor tissue had no significant difference in mRNA level and methylation level compared with adjacent non-cancerous tissues. One tumor tissue with higher methylation level had similar mRNA level with adjacent non-cancerous tissue. 2.2 5-Aza-CdR induces the changes of sall3 CpG island methylation levels and mRNA levels in HCC cell linesAfter treatment with 0.1,0.5,2.5μM 5-Aza-CdR, sall3 CpG island methylation levels were dose-dependently down-regulated and mRNA levels were dose-dependently up-regulated in Huh7, HepG2, SK-HEP1 and SMMC7721 cell lines; sall3 CpG island methylation levels were dose-dependently down-regulated but mRNA levels were irregularly up-regulated in Be17402 and QGY7703 cell lines.2.3 The methylatioin levels of sall3 CpG island in different clinicopathological factors in HCCThe methylation levels of sall3 CpG island were higher in female than in male and 3 CG units were significantly different;>48 years group exhibit higher methylation levels of sall3 than≤48 years group and 2 CG units were significantly different; HBV negative group had higher methylation levels than HBV positive group but all the 24 CG units were no significantly different. Methylation levels in many CG units were higher in moderate pathological phase than good phase and highest in poor phase, but most CG units were not significantly different.Conclusions1. Among the 38 samples,18 tumor tissues exhibited higher methylation levels of Oct6 CpG island than adjacent non-cancerous tissues. Among the 18 samples,17 had lower Oct6 mRNA levels than adjacent non-cancerous tissues and the methylaiton levels were negatively associated with mRNA levels (rs=-0.789, P< 0.001). In six HCC cell lines,5-Aza-CdR induced down-regulation of methylation levels of Oct6 CpG island and up-regulation of mRNA levels in HepG2% Huh7,Be17402,SK-HEP1,QGY7703 cell lines. The results suggest that DNA methylation is one of Oct6 regulatory mechanisms in HCC.2. Among the 38 samples,14 tumor tissues with lower Oct6 mRNA levels had similar methylation levels with adjacent non-cancerous tissues; in SMMC7721 cell line,5-Aza-CdR down-regulated Oct6 CpG island methylation levels, but the mRNA levels had no significant changes. The results suggest that Oct6 may exit other regulatory mechanisms in HCC.3. Among the 38 samples,27 tumor tissues exhibited higher methylation levels of sall3 CpG island than adjacent non-cancerous tissues. Among the 27 samples,24 had lower sall3 mRNA levels than adjacent non-cancerous tissues and the methylaiton levels were negatively associated with mRNA levels (rs=-0.714, P< 0.001).5-Aza-CdR induced down-regulation of methylation levels of sall3 CpG island and up-regulation of mRNA levels in all the six HCC cell lines (HepG2, Huh7, SMMC7721, Be17402, SK-HEP1, and QGY7703). The results suggest that DNA methylation is one of sall3 regulatory mechanisms in HCC.4. Among the 38 samples,9 tumor tissues with lower sall3 mRNA levels had similar methylation levels with adjacent non-cancerous tissues. The results suggest that sall3 may exit other regulatory mechanisms in HCC.