Study On Promoter Methylation Status Of Critical Functional Genes KISS1 And GnRH In GT1-7 Cell Line

Background: DNA methylation is an important style of epigenetic modification,which is characterized by a covalent addition of a methyl group mainly in the cytosine residues at position5 in Cp G dinucleotides,becoming 5-methylcytosine.DNA methylation plays an important role in maintaining normal cell function,embryonic development and human tumors,and it is one of the hot spots in the newest research.The Cp G island methylation of gene promoter region is an important way to inhibit the expression.The traditional DNA methylation research of clone sequencing is complicated in operation and expensive in price.The Next generation sequencing technologies has provided unprecedented opportunities for DNA methylation research.Objective: GT1-7 cell is an ideal model for the study of puberty.KISS1 gene and Gn RH gene are the two most important functional genes in the development of puberty.So far,The promoter methylation status of KISS1 gene and Gn RH gene has not been reported in the cell.This experiment using bisulfite treated the genome DNA of GT1-7 cells and L929 cells,then the PCR products used the next generation sequencing technology,at the same time,as the control experiment,the same batches of PCR products of GT1-7 cells use the Cloning sequencing.Methods: In GT1-7 cell line KISS1 gene has three transcripts,RT-PCR methods detect the expression of transcripts.PROMOTER2.0 and Neural Network Promoter are two common promoter prediction online software of Bioinformatics software,transcription factor also associate with gene promoter region,so we use JASPAR online predict transcription factor binding site in the promoter region.Bisulfite treated DNA.Then Methylprimer software design BSP primers for nested PCR,then use the next generation sequencing methods to detect promoter DNA methylation of GT1-7cells.Clone sequencing detect the positive clone plasmid of TA clone.Results: In GT1-7 cells,KISS1-002 transcript has the highest expression level of the three transcripts.In the promoter region of KISS1 gene and Gn RH gene DNA methylation information of 27 Cp G sites is complete.The next generation sequencing results show that the average methylation level of KISS1 and Gn RH is 78.74% and 88.09%.Clone sequencing results show thatmethylation level of KISS1 and Gn RH is 76.50% and 84.20%.And two sequencing methods results all have the information of 27 Cp G sites DNA methylation status.In L929 cells,the promoter region of KISS1 gene and Gn RH gene DNA methylation information of 27 Cp G sites is complete.The next generation sequencing results show that the average methylation level of KISS1 and Gn RH is 94.07% and 97.32%.Conclusion: The same results were detected in the detection of DNA methylation with the next generation sequencing and Clone sequencing.But the former methods each reads is equivalent to a monoclonal compared with the clone sequencing.so the next generation sequencing results are more accurate.