Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jan 15;10(1):16-39.
eCollection 2018.

Preeclampsia Is Associated With Hypermethylation of IGF-1 Promoter Mediated by DNMT1

Affiliations
Free PMC article

Preeclampsia Is Associated With Hypermethylation of IGF-1 Promoter Mediated by DNMT1

Min Ma et al. Am J Transl Res. .
Free PMC article

Abstract

Previous studies have demonstrated a dynamic epigenetic regulation of genes expression in placenta trophoblasts and a dynamic imbalance of DNA methylation and hydroxymethylation. Reduced IGF-1 has been observed in preeclampsia. This study was to investigate the interactive roles between IGF-1 and the global DNA methylation/hydroxymethylation, and the status of DNA methylation/hydroxymethylation and associated enzymes such as DNMTs and TETs in peeeclamptic placentas and hypoxic trophoblasts. It was found that IGF-1 was decreased in preeclamptic placentas and hypoxic trophoblasts when compared to the control group using immunohistochemisty, western blot, qRT-PCR and ELISA. Pyrophosphate sequencing showed IGF-1 promoter was significantly hypermethylated in preeclamptic placentas, which was responsible for reduced IGF-1 expression. Preeclamptic placentas and hypoxic trophoblasts were hypermethylated and hypohydroxymethylated accompanied by remarkably higher 5mC, DNMT1 and DNMT3b, and lower DNMT3a, 5hmC, TET1, TET2 and TET3 detected by immunohistochemisty, western blot, qRT-PCR and ELISA. Pearson's correlation confirmed a statistically significant negative correlation between IGF-1 and DNMT1. Furthermore, both treatment with 5-Aza-dc and DNMT1-siRNA significantly increased the expression of IGF-1 in HTR8 cells, indicating the potential mechanism of DNMT1-mediated DNA methylation in IGF-1 regulation. However, IGF-1 didn't change DNA methylation or hydroxymethylation. These findings suggest that preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1 and provide new insights into the diagnosis and treatment of preeclampsia.

Keywords: 5mC/5hmC; DNA methylation/hydroxymethylation; DNMTs; IGF-1; Preeclampsia; TETs.

Conflict of interest statement

None.

Figures

Figure 1
Figure 1
Reduced expression of IGF-1 in preeclamptic placentas. A. Immunohistochemistry revealed decreased levels of IGF-1 in PE group compared to NP group. B. Western blot showed lower expression of IGF-1 in PE group compared to NP group. C. qRT-PCR showed reduced levels of IGF-1 in PE group compared to NP group. Data were presented as Mean ± SD. **P<0.01, ***P<0.001. NP refers to normal pregnancy, PE refers to preeclampsia.
Figure 2
Figure 2
Decreased expression of IGF-1 in hypoxic HTR8 and JEG3 cells. A, D. ELISA showed decreased supernatants IGF-1 levels in hypoxic HTR8 and JEG3 cells, respectively. B, E. Western blot revealed reduced IGF-1 protein levels in hypoxic HTR8 and JEG3 cells, respectively. C, F. qRT-PCR depicted a reduction of IGF-1 mRNA levels in hypoxic HTR8 and JEG3 cells, respectively. Data were presented as Mean ± SD. *P<0.05, **P<0.01, ***P<0.001.
Figure 3
Figure 3
Hypermethylated IGF-1 promoter in preeclamptic placentas by pyrophosphate sequencing. A. Representative pyrosequencing images in NP and PE groups. B. The degree of DNA methylation of IGF-1 promoter at CpG_1 and CpG_2. Data were presented as Mean ± SD. **P<0.01. NP refers to normal pregnancy, PE refers to preeclampsia.
Figure 4
Figure 4
Preeclamptic placentas were hypermethylated with higher expression of 5mC, DNMT1 and DNMT3b, and lower expression of DNMT3a compared with normal pregnant placentas. A, B. Immunohistochemistry and ELISA showed increased 5mC levels in PE group compared to NP group. C-E. Immunohistochemistry, western blot and qRT-PCR revealed higher DNMT1 in PE group than NP group. F-H. Immunohistochemistry showed no significance of DNMT3a, while western blot and qRT-PCR depicted lower DNMT3a in PE group than NP group. I-K. Immunohistochemistry showed no significance of DNMT3b, while western blot and qRT-PCR depicted higher DNMT3b in PE group than NP group. Data were shown as Mean ± SD. *P<0.05, **P<0.01, ***P<0.001. NP refers to normal pregnancy, PE refers to preeclampsia.
Figure 5
Figure 5
Preeclamptic placentas were hypohydroxymethylated with lower expression of 5hmC, TET1, TET2 and TET3 compared to normal pregnancy. A, B. Immunohistochemistry and ELISA showed decreased 5hmC levels in PE group compared to NP group. C-E. Immunohistochemistry, western blot and qRT-PCR revealed lower TET1 in PE group than NP group. F-H. Immunohistochemistry, western blot and qRT-PCR revealed lower TET2 in PE group than NP group. I-K. Immunohistochemistry, western blot and qRT-PCR revealed lower TET1 in PE group than NP group. Data were shown as Mean ± SD. *P<0.05, **P<0.01, ***P<0.001. NP refers to normal pregnancy, PE refers to preeclampsia.
Figure 6
Figure 6
Hypoxic trophoblasts were hypermethylated with increased 5mC, DNMT1 and DNMT3b, and decreased DNMT3a compared to normoxic trophoblasts. A. Increased 5mC in hypoxic HTR8 and JEG3 cells. B. Increased DNMT1 in hypoxic HTR8 and JEG3 cells. C. Decreased DNMT3a in hypoxic HTR8 and JEG3 cells. D. Decreased DNMT3b mRNA levels from 24 h to 72 h and increased DNMT3b mRNA levels in 96 h in hypoxic HTR8 and JEG3 cells. Data were shown as Mean ± SD. *P<0.05, **P<0.01, ***P<0.001.
Figure 7
Figure 7
Hypoxic trophoblasts were hypohydroxyrmethylated with reduced 5mC, TET1, TET2 and TET3 compared to normoxic trophoblasts. A. Decreased 5hmC in hypoxic HTR8 and JEG3 cells. B. Decreased TET1 mRNA levels in hypoxic HTR8 and JEG3 cells. C. Decreased TET2 mRNA levels in hypoxic HTR8 and JEG3 cells. D. Decreased TET3 in hypoxic HTR8 and JEG3 cells. Data were shown as Mean ± SD. *P<0.05, **P<0.01, ***P<0.001.
Figure 8
Figure 8
IGF-1 was negatively correlated with 5mC and DNMT1, and positively correlated with 5hmC, TET1, TET2 and TET3. A. IGF-1 and 5mC: R=-0.496, P=0.026. B. IGF-1 and DNMT1: R=-0.475, P=0.034. C. IGF-1 and DNMT3a: P=0.178. D. IGF-1 and DNMT3b: P=0.683. E. IGF-1 and 5hmC: R=0.471, P=0.036. F. IGF-1 and TET1: R=0.764, P<0.001. G. IGF-1 and TET2: R=0.529, P=0.016. H. IGF-1 and TET3: R=0.587, P=0.007.
Figure 9
Figure 9
IGF-1 was upregulated caused by its hypomethylated promoter region in HTR8 cells treated with 5-Aza-dc and DNMT1-siRNA. A, B. Increased IGF-1 in HTR8 cells treated with 4µM 5-Aza-dc. C, D. Increased IGF-1 in HTR8 cells treated with DNMT1-siRNA. E. Representative image of DNA integrity verified by agarose gel electrophoresis. F. IGF-1 promoter region was hypomethylated at CpG_1 and CpG_2 in HTR8 cells treated with 5-Aza-dc and DNMT1-siRNA. G. Representative pyrosequencing images of IGF-1 in four groups. Data were shown as Mean ± SD. *P<0.05, **P<0.01, ***P<0.001. NC refers to negative control.
Figure 10
Figure 10
Upregulation of IGF-1 didn’t alter genomic DNA methylation. A. IGF-1 was upregulated by pEX-2-IGF-1. B. Upregulation of IGF-1 didn’t alter DNMT1 expression. C. Upregulation of IGF-1 didn’t alter DNMT3a expression. D. Upregulation of IGF-1 didn’t alter DNMT3b expression. E. Upregulation of IGF-1 didn’t alter 5mC levels. F. Upregulation of IGF-1 didn’t change TET1 expression. G. Upregulation of IGF-1 didn’t change TET2 expression. H. Upregulation of IGF-1 didn’t change TET3 expression. I. Upregulation of IGF-1 didn’t alter 5hmC levels. Data were shown as Mean ± SD. *P<0.05, **P<0.01, #P<0.05, ##P<0.01.
Figure 11
Figure 11
Downregulation of IGF-1 didn’t change genomic DNA hydroxymethylation. A. IGF-1 was downregulated by IGF-1-siRNA. B. Downregulation of IGF-1 didn’t change DNMT1 expression. C. Downregulation of IGF-1 didn’t change DNMT3a expression. D. Downregulation of IGF-1 didn’t change DNMT3b expression. E. Downregulation of IGF-1 didn’t change 5mC levels. F. Downregulation of IGF-1 didn’t alter TET1 expression. G. Downregulation of IGF-1 didn’t alter TET2 expression. H. Downregulation of IGF-1 didn’t alter TET3 expression. I. Downregulation of IGF-1 didn’t alter 5hmC levels. Data were shown as Mean ± SD. *P<0.05, **P<0.01, ***P<0.001. NC refers to negative control.

Similar articles

See all similar articles

Cited by 8 articles

See all "Cited by" articles

LinkOut - more resources

Feedback