Abstract
OBJECTIVE DNA methylation has been regarded as an important epigenetic signature reflecting the transcription state of DNA in cells. This study was to conducted to assess the relationship between human alpha-fetoprotein (AFP) gene expression and the DNA méthylation status of the promoter region in three different cells, namely two human hepatocellular carcinoma (HCC) cell lines and normal human fibroblasts.
METHODS Transcription of the AFP gene was verified by RT-PCR. After bisulphate treatment of DNA, the methods of MSP and BSP were used to analyze the méthylation density and status within single DNA strands of two closely spaced CpG dinucleotides of the promoter region in the different cells.
RESULTS RT-PCR analysis indicated that the expression of the AFP gene in HepG2 cells was significantly higher than in SMMC-7721 cells, and that the AFP gene was not expressed in normal human fibroblasts. By MSP and BSP we observed that the promoter region was demethylated in the AFP-high-expressing cell lines, and that the sites of -2,494 bp and -2,431 bp in the AFP genomic sequence can be used as detection sites for early tumorous diagnosis.
CONCLUSION These results indicate that the DNA méthylation state of the promoter region has a negative correlation with AFP gene expression.
keywords
Cancer, diabetes, and vascular disease are three major human health afflictions. Among those maladies, cancers have the greatest effect on survival and the patients' quality of life. As we know, early detection and therapy by surgical resection or chemotherapy are very important to achieve positive results. Hence, people are more and more concerned about early and accurate tumorous diagnosis.
AFP is a Mr 70,000 glycoprotein, produced at high levels by fetal liver, which is transcriptionally repressed after birth. Due to aberrant AFP gene expression that is characteristic of tumors related to the fetus or liver, serum AFP has been used as a diagnostic marker for these tumors.111 Although AFP has been extensively studied, little is known about the control of AFP expression.
Allele-specific méthylation plays an important role in regulating expression of the AFP gene in a wide range of eukaryotes.[2] In our study, RT-PCR was applied to identify differentially expressed AFP genes among three different cell lines. DNA méthylation status of the AFP promoter region was examed by MSP and BSP.
MATERIALS AND METHODS
Cell culture
The human hepatocellular carcinoma HepG2 and SMMC-7721 cell lines were maintained in high glucose Dulbecco' modified Eagle's medium (DMEM) and RPMI 1640 medium, respectively. All media were supplemented with 10% fetal bovine serum (¡FBS), 100 IU/ml penicillin and 100 |xg/ml streptomycin (PS). The human fibroblasts were maintained in low glucose DMEM medium supplemented with 15% FBS and PS.
Reverse transcriptase polymerase chain reaction
Total RNA was extracted from the cultured cells with the Trizol reagent (Invitrogen, USA). The cDNA was synthesized from RNA using avian reverse transcriptase (GIBCO BRL, Life Technologies, USA) and random hexamers (TaKaRa Biotechnology, Japan) in a 25 /xi volume by means of incubation at 43°C for 60 min, then stored at -20°C until used for PCR. The cDNA was amplified by 35 cycles of PCR. The sequences of primers used in the experiment are shown in Table 1. Five p.1 of cDNA solution was mixed with 45 p.1 of PCR reaction buffer containing 25 p.1 Premix Tag polymerase (TaKaRa, Japan), and 25 pmol of each primer. The amplification was performed in a DNA Thermal Cycler (Biometra UNOII, USA). After 5 min at 94°C, the reaction mixture was subjected to 35 cycles of the PCR program set (denature at 94°C for 30 s, anneal at 50°C for 45 s, extend at 72°C for 45 s). The reaction was terminated at 72°C at 5 min. Six p.1 of above amplified products were subjected to electrophoresis on 1.5% agarose gels, stained with ethidium bromide, detected by UV light and photographed with a gel imaging analysis system.
Primers and conditions used
Preparation of DNA and detection by PCR and sequencing
The genomic DNA was prepared using a Classic Genomic DNA Isolation Kit, and stored in TE buffer at -20°C until used. The templates included genomic DNA and products after digestion with different enzymes. Two pi of DNA solution was mixed with 18 μ of PCR reaction buffer containing 10 μ Premix Tag polymerase, and 25 pmol of each primer (Tablel). After 3 min at 94°C, the reaction mixture was subjected to 34 cycles of the PCR program set (denature at 94°C for 30 s, anneal at 50°Cfor 30 s, extend at 72C for 30 s). The reaction was terminated at 72°C at 10 min. After the products were identified by electrophoresis, representative bands from the AFP gene were gel-purified, and followed by automatic DNA sequencing.
Bisulphate treatment of DNA
After digestion of genomic DNA with different enzymes, sodium bisulfite was used to modify the genomic DNA fragment. The products were purified using a Promega Wizard DNA Purification System to remove excess sodium bisulfite, then the DNA was precipitated overnight to obtain the modified DNA. The modified DNA samples were finally stored at -20°C for further use.
Design of the primers
The primer pairs (Table 1) for MSP and BSP analysis were designed with the primer design software on line (http://microgen.ouhsc.edu/cgibin/primer3_www.cgi).
Methylation specific PCR (MSP)
Two μl of modified DNA solution was mixed with 18 μ of PCR reaction buffer containing 10 μl HotStart Tag polymerase PCR MasterMix (Tiangen, Beijing), and 25 pmol of each primer (including Methylation primers and unmethylation primers). The amplification was performed in a DNA Thermal Cycler (Biometra UNOII, USA). After 4 min at 94°C, the reaction mixture was subjected to 15 cycles of the PCR program set (denature at 94°C for 30 s, anneal at 61°C for 30 s, extend at 72°C for 30 s), then the reaction mixture was subjected to 25 cycles of the PCR program set (denature at 94°C for 30 s, anneal at 60°C for 30 s, extend at 72°C for 30 s). The reaction was terminated at 72°C at 5 min. MSP products were identified by electrophoresis.
Bisulfite sequencing PCR (BSP)
At first, 2 μl of modified DNA solution was mixed with 17.6 μl of PCR reaction buffer containing 2.5 μl of 2.5 mM dNTP and 25 pmol of each primer. After 7 min at 97°C, 1 unit of Tag polymerase was added to the PCR reaction system. Then, after 3 min at 94°C, the reaction mixture was subjected to 10 cycles of the PCR program set (denature at 94°C for 30 s, anneal at 60°C < each cycle decrease for 0.5°C > for 30 s, extend at 72°C for 30 s), the reaction mixture was subjected to 5 cycles of the PCR program set (denature at 94°C for 30 s, anneal at 54°C for 30 s, extend at 72°C for 30 s), the reaction mixture was subjectèd to 5 cycles of the PCR program set (denature at 94°C for 30 s, anneal at 53°C for 30 s, extend at 72°C for 30 s), the reaction mixture was subjected to 10 cycles of the PCR program set (denature at 94°C for 30 s, anneal at 52°C for 30 s, extend at 72°C for 30 s), the reaction mixture was subjected to 15 cycles of the PCR program set (denature at 94°C for 30 s, anneal at 51°C for 30 s, extend at 72°Cfor 30 s). The reaction was terminated at 72°Cat 5 min. After the BSP products were identified by electrophoresis, to verify the PCR results, representative bands from the AFP gene were gelpurified, followed by automatic DNA sequencing, contrasting the Methylation sites.
Results
Cell morphology
The hepatocellular carcinoma cells showed derangement of size, the karyoplasmic ratio was inverted and there was a loss of normal contact inhibition. After filling the flask, they overlaped, and showed signs of infinite poliferation. The human normal fibroblasts showed fusiform, and arranged as swirls.
Total RNA
Preparation. To avoid contamination and degradation of RNA, we used DNAase I. RNA samples including OD260/280 > 1.8 and showing three clear bands at 5,000 bp, 2,000, bp and 300 bp with formaldehyde degeneration gel electrophoresis were selected.
The expression of the AFP mRNA in the three kinds of cells was different (Fig. 1). With electrophoresis, products of the HepG2 cells were observed as clear bands at 451 bp and at 350 bp; products of the SMMC-7721 cells were observed as a fuzzy band at 451 bp and a clear band at 350 bp; but products of the human fibroblasts were observed a clear band at 350 bp. Length of the fragments were in coincidence with the design. The 2D image display of the AFP gene expression in HepG2 cells was 10 times that of the SMMC-7721 cells using β-actin as inner reference by the gel imaging analysis system. Results from the human fibroblasts were negative, indicating that the experimental results were precise and specific.
RT-PCR products analyzed by agarose gel electrophoresis. AFP gene expression in HepG2 (1,3) and SMMC-7721 (4,6) and fibroblasts (7,9), ß-actin gene expression in HepG2 (2,3) and SMMC-7721 (5,6) and fibroblasts (8,9). M: 100~600bp
Preparation of genomic DNA and detection by PCR and sequencing
With electrophoresis, genomic DNA of the three kinds of cells can be observed as a clear band at 23,130 bp using X.-HindIII digest DNA Marker as inner reference. Setting up PCR amplification reactions with different template DNA was to identify whether enzymatic digestion had an influence on the objective fragments of the genomic DNA. The PCR products can be observed as a clear band at 269 bp (Fig.2). The sequencing results of the PCR products were in coincidence with the design (Fig.3).
PCR results of the original DNA in the AFP promoter region of HepG2 (1,2), SMMC-7721 (3,4) and fibroblasts (5,6)1,3,5: genomic DNA template; 2,4,6: DNA after digestion by enzyme; M: 100~600bp.
Part of the sequencing result of HepG2 (upper) and fibroblasts (inferior) in the promoter region with original DNA.
The results of BSP
With electrophoresis, products from the three cells were all observed as clear bands at 248 bp(Fig.5). The length of the fragments was in agreement with the design. Sequencing results (Fig.6) were analyzed by DNAssist 2.0 software. We can deduce that the unmethylated CG status was normal in HepG2 cells, the methylated CG status of SMMC-7721 cells was higher than the HepG2 cells, but the methylated CG status in fibroblasts was the highest. Two CG sites where the methylated incidence was higher in the hepatoma carcinoma cells compared to fibroblasts can be used as detection sites for early tumorous diagnosis. They were located in the AFP genomic sequence -2494bp and -2431bp. Because there was a difference between the HepG2 cells and SMMC-7721 cells in methylated CG status, we suggest that the two CG sites have corelations with AFP expression in the two kinds of hepatoma carcinoma cells.
MSP results with the unmethylated primer (1,3,5) and methylated primer (2,4,6) in the AFP promoter region of HepG2 (1,2), fibroblasts (3,4) and SMMC-7721 (5,6) M:50bp~500bp.
BSP results of the AFP promoter (1,2,3) region in HepG2 (1),fibroblasts (2) and SMMC-7721 (3) M: 50bp~500bp
Part of the sequencing analysis of HepG2 (upper), SMMC-7721 (middle) and fibroblasts (inferior) in the promoter region with BSP.
DISCUSSION
The albumin gene family is comprised of four genes encoding albumin, alpha-albumin (ALF), AFP, and vitamin D-binding protein, which are tandemly linked in the 4q sub-centromeric region.[3] Among them, AFP is selectively expressed in fetal liver, while expression of ALF and vitamin D-binding protein is restricted to newborn and adult liver.[4] However, AFP expression appears to be reactivated in 60 ±70% of hepatocellular carcinoma (HCC) cells, and stimulation of hepatoma cell growth is believed to be one of the biological functions of AFP.[5] In humans, two major clusters of the AFP gene are localized on chromosomes lip 15.5 and 15ql 1 -13.[6]
In studies on the control of gene expression, much recent work using the methods of genetic engineering has suggested that DNA Methylation (on some cytosines) plays a role in the control of gene activity. Undermethylation is generally accompanied by gene expression. A review of studies on DNA Methylation in some physiopathologie conditions (such as development and cancer) and of recent therapeutic trials related to beta thalassemia, allows one to conclude that there is a correlation between the DNA Methylation pattern and gene expression, rather than a direct causal effect.[7]
In 1982, Andrews et al.[8] showed that 6 CCGG sites of the AFP gene had a negative relationship with its expression. This study started research, to analyze the DNA Methylation pattern of the AFP gene and its expression. After that, a number of investigations demonstrated that there was a correlation between the DNA Methylation pattern and gene expression. But the methods were restricted to hybridizing labeled cDNA clones to Hpall and Ms[9] These CCGG-cutting enzymes distinguish 5-methylcystosine in mCCGG (sensitive to Hpall) and CmCGG (sensitive to MspI). Based on this practice, the method always showed a false positive rate and less sites could be detected. Of most importance, it could not display the original methylated status.
In 1992, a procedure for the analysis of the Methylation status was described. The method offers a rapid and reliable alternative to conventional methods. The efficient resolution of the differentially methylated alleles is demonstrated for genes. The method is based on MSP [10] and BSP. Briefly, genomic DNA is initially subjected to an in vitro bisulfite treatment, whereby unmethylated cytosines are deaminated. Subsequent PCR amplifications, using primers specific for modified DNA, are aimed at DNA segments that show parent-of-origin-specific Methylation. [11] PCR conditions are chosen that allow àn efficient amplification of both alleles. The PCR products representing the two alleles are identical in size; they differ, however, at a number of positions within the amplified DNA segment. We observed that, in the promoter area, the Methylation density negatively modulated with expression of AFP. The two CG sites of the promoter area where the Methylation degree was higher in hepatoma carcinoma cell than in fibroblasts can be used as a detection sites for HCC. These are sites at -2,494 bp and -2,431 bp in the AFP genomic sequence.
In conclusion, our data indicate that AFP gene expression, through epigenetic changes and allele loss, is a common and important event in the carcinogenesis of malignant liver tumors.[12]
Footnotes
This work was supported by the Tianjin Municipal Science and Technology Commission(No.05YFJMJC08100).
- Received June 29, 2006.
- Accepted September 14, 2006.
- Copyright © 2006 by Tianjin Medical University Cancer Institute & Hospital and Springer
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