Study Of Edneb Gene's Silencing Induced By Methylated Oligonucleotides In Hirschsprung Disease's Pathogenesis

PartⅠSilencing of EDNRB Gene Induced by Methylated Oligonucleotides in Mouse Embryonic Stem CellAims:Hirschsprung's disease (HD) is the most common intestinal malformations of Pediatric Surgery. Abnormality of EDNRB/EDN3/ECE1 signal transduction way is confirmed as the most common pathogenesis etiology of HD, but the mutations coul dn't account for all HD and the EDNRB's mutation rate is no more than 3%. This study is to design and synthesize specific methylated oligonucleotides (MON) comp lementary with EDNRB gene's promoter and elucidate the correlation between EDN RB gene promoter's methylation and HD's pathogenesis.Methods:Mouse EDNRB gene's DNA and promoter's sequences were retrieved fro m Genbank and DBTSS. Specific methylated oligonucleotides complementary with E DNRB gene's promoter were designed following the criteria and marked with FITC in 5'. Effectine were used to transfect the MON into C57BL/6 embryonic stem eel 1 (mESC) and then separated with flow cell sorter.5-Aza-2'-deoxycytidine (5-Aza-dC) were used for demethylation. Bisulfite genomic sequencing (BSP), real-time quantit ative RT-PCR (qRT-PCR) and Western blot were applied to detect the promoter me thylation change, mRNA and protein expression of EDNRB gene pre-and post-trans fection and 5-Aza-dC treatment, respectively.Results:1008～2889bp in Mouse EDNRB gene's promoter was a CpG island:A%: 23.22, T%:23.65,C%:25.13,G%:28.00,C+G%:53.13,CpG%:4.20,A+T/C+G%:0.88. The t ransfection efficiency of MON by Effectine was 40.2%. MON1 was complementary with PCR product's 3,4,5CG. The methylation rate of 1～16 CGs before transfecti on were all 0%, but it changed to 30%,40%,60%,60%,60%,50%,40%,40%,40%,40%, 40%,40%,30%,20%,20% and 10%, and they all decreased to 0% after 5-Aza-dC trea tment. MON2 was complementary with PCR product's 9,10,11CG. The methylatio n rate of 1～16 CGs before transfection were all 0%, but it changed to 20%,30%, 40%,40%,50%,40%,50%,60%,70%,70%,70% 70%,60%,40%,40% and 30%, and they al 1 decreased to 0% after 5-Aza-dC treatment. MON3 was complementary with PCR product's 13,14,15CG. The methylation rates of 1～20 CGs before transfection we re all 0%, but it changed to 10%,10%,30%,30%,30%,40%,50%,50%,50%,60%,60%,6 0%,70%,70%,70%,60%,60%,50%,30% and 10%, and they all decreased to 0% after 5-Aza-dC treatment. MON4 was complementary with PCR product's 2,3,4,5CG. T he methylation rate of 1～8 CGs before transfection were all 0%, but it changed t o 10%,20%,20%,20%,20%,10%,10% and 0%, and they all decreased to 0% after 5-Aza-dC treatment. The methylation rates of CON1 and CON2 in EDNRB gene pro moter pre- and post-transfection and 5-Aza-dC treatment was all 0%. The EDNRB/ GAPDH (mRNA/mRNA) and EDNRB/β-actin (Protein/Protein) before MON1, MON 2, MON3, MON4, CONland CON2 transfection in C57BL/6 showed no differences. EDNRB/GAPDH and EDNRB/β-actin decreased after MON1, MON2, and MON3 t ransfection. They all increased to normal level in MON1, MON2, and MON3 transf ection mESC after 5-Aza-dC treatment. Conclusion:Specific complementary methylated oligonucleotides could induce the m ethylation of EDNRB gene's promoter. The ability of methylation induction and exp ression inhibition may correlate with MON's complementary CG sites in the targete d gene promoter. PartⅡInheritance of EDNRB gene promoter's methylation induced by methylated OligonucleotidesAims:Methylation as the most important regulation in Epigenetics is characteristic of stable genetic inheritance, which could be inherited in generation-generation and cell cycle. The methylated DNA strand formed hemi-methylated replication fork during mitosis, which could form substrate for DNA Methyltransferase. This semiconservative replication mechanism ensures the stable inheritance of methylation pattern. This study is to design and synthesize specific methylated oligonucleotides (MON) complementary with EDNRB gene's promoter and study its inheritance stability.Methods:Mouse EDNRB gene's DNA and promoter's sequences were retrieved from Genbank and DBTSS. Specific methylated oligonucleotides complementary with EDNRB gene's promoter were designed following the criteria and marked with FITC in 5'. Effectine were used to transfect the MON into C57BL/6 embryonic stem cell (mESC) and then separated with flow cell sorter. The separated cells were cultured for generations. Bisulfite genomic sequencing (BSP), real-time quantitative RT-PCR (qRT-PCR) and Western blot were applied to detect the promoter methylation change, mRNA and protein expression of EDNRB gene pre- and post-transfection and culture for generations.Results:The mean methylation rate after MON1, MON2, MON3 and MON4 transfection were 60%,70%,70% and 20%, respectively. The mean methylation EDNRB gene promoter after MON1, MON2 and MON3 transfection decreased as culture generation's increasing and they became identical in the 6th generation, while it was 3rd generation in after MON4 transfection. The mRNA and protein expression of EDNRB gene after MON1, MON2 and MON3 significantly decreased and it increased as culture generation's increasing. MON1, MON2 and MON3 gene expression became identical in the 6th generation, while it was 3rd generation after MON4 transfection.Conclusion:The methylation of EDNRB gene's promoter induced by methylated oligonucleotides couldn't be inherited stalely; it can decrease as culture generation's increasing with progressive recovered gene expression. It couldn't establish a mouse HD model by methylation of EDNRB gene's promoter induced by methylated oligonucleotides.