The Methylation Status Of AFP Gene And It's Expression In Vivo

Objective: The abnomal status of DNA methylation take an important role in the development of cancer. In this study, we try to explore the correlation between the methylation status of Alpha-fetoprotein (AFP) gene in the promoter region and it's expression.Methods:1 Cell culture: the human hepatocellular carcinoma cell line (MHCC97_H) are subcultured to be isolated the DNA and RNA from them.2 Orthotopic transplanted nude mice: six male nude mice transplanted in their left liver with human hepatocellular carcinoma tumor and two without the transplantion were bred in the circumstance of SPF and killed six weeks later. Pathology samples,total protein,DNA and RNA were prepared from the hepatoma tissue.3 Assessment of the animals: The genomic DNA were prepared from the cells and tissues and modified by the Sodium bisulfite. AFP gene fragments are amplificated (the genomic DNA is the template ). The sequence of the ptoducts are detected automatic DNA sequencing and contrasted with the human genomic DNA.4 WESTERN BLOT: Solubilized protein samples from the MHCC97_H cell line,human fibroblast cell line and hepatoma tissue are firstly separated by using SDS-PAGE,and then electrophoretically transferred to a nitrocellulose membrance. After blocking with a protein blocking agent , the membrance is probed with the primary antibody. The antibody-antigen complexes are then identified by the secondary anti-IgG antibody, which is conjugated to horseradish peroxidase(HRP) enzymes.Finally, chromogenic substrates are used to visualize the activity. In the whole procedure , theβ-actin gene was used as the inner reference to find the difference in the espression of the AFP from different speciments.5 The primer spairs for MSP and BSP are designed online (http://www.humgen.nl/primer_design.html) , the others by the Primer Premier 5.06 RT-PCR: RNA are prepared from the cells and tissue by the TRIzol and reverse transcribed into CDNA, which is the template in the amplification of the AFP andβ-actin gene (as the inner reference in the procedure )fragment .7 Bisulphate treatment of the DNA: After cleaned up by using Wizard DNA Purification System, the genomic DNA were stored at -20℃.8 The methylation density of promoter region in the human AFP gene from cells and tissue analyzed with Methylation Specific Polymerase chain reaction method (MSP): AFP gene fragment is amplificated(MoDNA is the template) by MSP primers (including methylation primers and unmethylation primers)and it's products was detected by electrophoresis.9 The methylation status within single DNA strands of two closely spaced CpG dinucleotides of promoter region in the human AFP gene from cells and tissue are analyzed with bisulfite sequencing PCR (BSP): AFP gene fragments are amplificated(MoDNA is the template) by BSP primers. The products are detected by electrophoresis and gel-purified , followed by automatic DNA sequencing, contrasting the methylation sites.Results:1 Morphology observation: The hepatocellular carcinoma cells were derangement inequality of size, karyoplasmic ratio inversion and losing normal contact inhibition. After filled the bottle, they became overlap, has the ability of infinite breeding.2 Orthotopic transplanted nude mice: First, 5×106～5×107cells were syringed into the right back of a Balb/c mouse.One week later,there is a tumor looks like a soybean being.Second,the rumor was removed from the body and dissected into several small ones which would be transplanted into the livers of the mice.When transplanted, the mice were paralysed sterily with the Barbital Sodium in the abdomen.Four weeks later after operation, there is a nugget like a soybean in the liver region of the nude mouse when you touch it's abdomen. He didn't like moving, but sleeping. Their skins were rough and shineless. With the time passed over , the tumors growed larger and larger. Sometime, it is so large that it look like a bean swelling up attached the skin in the bellies.3 Assessment of the animals: It is found that the sequence of the DNA from the cancer in the nude mice consisitent with the human AFP gene by blasting it in the NCBI.4 Western Blot: Solubilized protein samples from the MHCC97_H cell line,fibroblast cell line and hepatoma tissue are detected by using Western Blot. It show that there is the AFP in the protein samples from the MHCC97-H cells and hepatoma tissue, but not in the fibroblast cells.5 RT-PCR:The RNA with OD260/280>1.8 was amplified. By electrophoresis, there ia a clear strap at 451bp and 350bp in the PCR product of cDNA from MHCC97_H cells and animal tissue. But only the strap at 350bp in the fibroblast. The length of the fragments were coincidence with which have been designed. All the results indicate the experimental results are specific and creditable.6 The result of MSP: There is a clear strap at 150bp amplified by unmethylation primer and a fuzzy trap at 142bp amplified by methylation primer in the PCR product of the MoDNA from MHCC97_H cells and tissue, but the opposite in the fibroblasts cells. From this result, it can be shown that there is a lower methylation density in the promoter of the AFP gene from the MHCC97_H cells and tissue.7 The result of BSP: By electrophoresis, there is a clear strap at 248bp in the product of BSP. The length of the fragment was coincidence with which have been designed. By the sequencing and aligning with DNAssist 1.0, we can get the conclusion that there is a lower degree of methylation in the promoter of AFP gene from MHCC97-H cells and animal tissue than the normal. That indicate the correlation between the methylation status of AFP gene and it's expression. Two CG sites of promoter region influence the expression of AFP gene.Conclusion:1 The human AFP gene expressed in hepatoma carcinoma cell and hepatoma tissue of the orthotopic transplanted nude mice ,but not in the normal cells and tissues.2 In promoter area, the methylation density negatively modulates expression of AFP activity.3 Two CG sites of promoter area where the methylation degree lower in hepatoma carcinoma cell and hepatoma tissue than fibroblast can be used as detection sites of HCC, which were at the site of -2494bp and -2431bp in AFP genomic sequence.