Relationship Between Gene Expression And Promotor Methylation Of HBs And HBx In Early Embryonic Cells

[BACKGROUND & AIM]Hepatitis B is a public health problem worldwide. The world governments and scientists pay particular attention to the transmission, pathogenic mechanism, methodology of diagnosis and treatment of HBV infection. It has been demostrated that HBV DNA could be integrated into human sperm chromosomes and that the sperm mediated HBV genes are able to replicate themselves and express their function in early embryonic cells. However, its molecular mechanism is largely unknown. DNA methylation is one of important aspects in epigenetic modifications and plays an important role in regulation of early embryo development. It also is a very important manner for cells to regulate the expression of host genes and viral genes entering into cells. HBV infection could cause epigenetic abnormality of host cells, induce the chromosomal instability which result in embryo abnormal development. The integration of HBV DNA into host genome could increase the risk of primary hepatocellular carcinoma. The aim of the present study is to investigate the relationship between gene expression and promotor methylation of HBV in early embryonic cells by study of HBV DNA methylation in the genome of sperm and embryonic cells.[MATERIALS AND METHODS](1) Materials:①the recombinant plasmid pBR322-HBV.②the 6-8 week old female golden hamsters.③sperm samples from HBsAg-negative volunteers.④methylation kit (Chemicom Company.S7820). (2) Methods:①transfection method: human sperm transfected with plasmid pBR322-HBV.②PCR and Dot blot methods: to detect if human sperm carry HBV DNA.③the methods for interspecific in vitro fertilization between human sperm and zona-free hamster ova and post culture: to obtain the early embryonic cells.④MSP method: to detect the methylation states of promoters of HBs and HBx genes in sperm and early embryonic cells.⑤BSP and BLAST analysis: to detect the methylation rate of HBs gene promoter.⑥RT-PCR: to detect the expression of HBs and HBx genes in the early embryonic cells. [RESULTS]①Dot hybridization showed negative results in the last four of six washing solutions after exposure of human sperm to pBR322-HBV, which eliminated the possibility of false positive due to the contamination of washing solutions.②PCR results showed sperm and embryonic cells contained HBV DNA sequences.③one cell- and two cell- embryos were obtained by interspecific in vitro fertilization between human sperm and zona-free hamster ova and post culture.④MSP result indicated that the promotors of HBs and HBx genes were unmethylated in pBR322-HBV and were methylated in sperm. However, they were unmethylated again in one cell- and two cell- embryos.⑤BSP and BLAST analysis showed the 112 bp positive band in HBs promoter region from sperm genome sample. All the CpG sites in that region were methylated.⑥RT-PCR results demonstrated the expression of HBx and HBs genes in the early embryonic cells.[CONCLUSION]①The promoter regions of HBs and HBx genes were found to be unmethylated in recombinant plasmid pBR322-HBV.②The promoter regions of HBs and HBx genes were found to be methylated in the sperm genome trasfected with pBR322-HBV.③The promoter regions of HBs and HBx genes were found to be unmethylated in the one-cell and two-cell embryos.④Sperm mediated HBx and HBs genes were expressed at mRNA level in the one-cell and two-cell embryos.⑤The expression of HBx and HBs genes were related to the unmethylation of their promoter regions in early embryonic cells.