title: [Study on the methylation of LCHAD gene promoter region in mitochondria of trophoblast cells incubated with long-chain fatty acids].
abstract: OBJECTIVE
  To explore the methylation level of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) gene promoter region in mitochondria of trophoblasts incubated with long-chain fatty acids and the time-effect of methylation modification.
  
  
  METHODS
  Primary human trophoblast cells and HTR8/Svneo cells were incubated with free fatty acids of various lengths. Long-chain free fatty acids (LC-FFA) was experimental group, short-chain fatty acids (SC-FFA) and medium-chain fatty acids (MC-FFA) were control groups, and blank control was without free fatty acid. Collecting cells and extract DNA at 24, 48 and 72 h incubation respectively. Predicted CpG island location access to 2 000 bp DNA sequences upstream of the transcription start site of LCHAD gene. We designed methylation detection sites and primer originally. Methylation of CpG sites in LCHAD gene promoter region were detected by MassARRAY and analyzed statistically.
  
  
  RESULTS
  (1) We detected methylation of 65% (11/17) CpG sites, including 8 single sites and 3 composite sites, in ampliconic sequences. These CpG sites were at positions: -984, -960, -899, -853, -811, -796, -774, -727, -615, -595, -579, respectively. In different groups methylation level and changes of every site showed differences with the most significant changes at -899 site. (2) Methylation of CpG island in LCHAD gene promoter region: ① Methylation of CpG island in LC-FFA and MC-FFA groups showed rising trend with the time: Methylation level of LC-FFA group at 72 h (0.55±0.08) was significantly higher than that of 48 h (0.35±0.12) and 24 h (0.31±0.04) (P<0.05). Methylation level of MC-FFA group at 72 h (0.44±0.05) was significantly higher than that of 24 h (0.31±0.04) (P<0.05). ② Methylation of CpG island in LCHAD gene promoter region in different groups at different times: Methylation level of LC-FFA group at 72 h was significantly higher than that of the other two groups at 72, 48 and 24 h (P<0.05). (3) Methylation of 11 CpG sites in LCHAD gene promoter region in different groups at 72 h: Methylation level of -899 site in LC-FFA and MC-FFA groups were significantly higher than that of other sites (P<0.05). (4) Methylation level of -899 site in LCHAD gene promoter region in LC-FFA group showed rising trend with the time: (72 h (0.34±0.15), 48 h (0.14±0.05) and 24 h (0.10±0.02), P<0.05). Methylation level of LC-FFA group at 72 h was significantly higher than that of the other two groups at 72, 48 and 24 h (P<0.05).
  
  
  CONCLUSIONS
  Methylation modification effect on LCHAD gene promoter region in trophoblast cells incubated with long-chain fatty acids is more significant than with medium-chain and short-chain fatty acids and shows obvious time-effect as incubation time prolonged. The changes at -899 site dominate the degree of methylation in LCHAD gene promoter region.
authors.affiliation: Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China.
authors.forename: Ran
authors.initials: R
authors.lastname: Meng
authors.affiliation: Email: zi_yang2005@126.com.
authors.forename: Zi
authors.initials: Z
authors.lastname: Yang
authors.forename: Hailing
authors.initials: H
authors.lastname: Wang
authors.forename: Yanling
authors.initials: Y
authors.lastname: Wang
authors.forename: Fengqiu
authors.initials: F
authors.lastname: Li
authors.forename: Yiwei
authors.initials: Y
authors.lastname: Han
mesh_terms: D002478:Cells, Cultured; D019175:DNA Methylation; D005227:Fatty Acids; D006801:Humans; D063989:Long-Chain-3-Hydroxyacyl-CoA Dehydrogenase; D008928:Mitochondria; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D014327:Trophoblasts
chemical_list: D005227:Fatty Acids; D063989:Long-Chain-3-Hydroxyacyl-CoA Dehydrogenase