Figure 1.
Schematic diagram of combined bisulfite restriction analysis (COBRA) of LINE-1.
A. Intact LINE-1 is approximately 6 kb long, and comprised of 5′-untranslated region (5′UTR), two opening reading frames ORF1 and ORF2 and 3′UTR. After bisulfite conversion of genomic DNA, PCR was performed to amplify a region of 160 bp in 5′UTR (gray region with oblique lines) harboring 10 CpG sites (marked by asterisks). The site at 62 bp is cut by Tsp509I (target at ∧AATT) when CpG is unmethylated, yielding fragments of 62 bp and 98 bp (marked as unmet). The site at 80 bp is cut by TaqαI (target at T∧CGA) when CpG is methylated, yielding two fragments of 80 bp each (marked as met). B. After digestion with restriction enzymes, PCR products were separated by electrophoresis in 2.3% agarose gels. DNA bands were visualized and subject to densitometry analyses. The LINE-1 methylation level is calculated as the percentage of the intensity of TaqαI-positive fragment in total DNA.
Table 1.
Primers used in real-time quantitative PCR.
Figure 2.
Assessment of LINE-1 methylation of first (1N) and third trimester (3N) placentas by COBRA.
A. A representative gel picture of LINE-1 COBRA showing the DNA bands for methylated (80 bp) and unmethylated LINE-1(98 bp and 62 bp). Lanes 1–5: 1N placental samples; Lane 6–10: 3N placental samples. B. The results of densitometry analyses on LINE-1 methylation. LINE-1 is hypomethylated in 3N placentas (Mean: 30.2%) relative to 1N placentas (Mean: 59.3%). * P<0.05. C. Inverse correlation between LINE-1 methylation levels with gestation age. W: weeks.
Figure 3.
Results of LINE-1 bisulfite sequencing.
Bisulfite converted DNA sequences and a typical sequencing result is shown at the top panel. Asterisks indicate the CpG sites. The cutting sites for Tsp509I and TaqαI were marked. 15 clones representing five first trimester (1N, left penal) placentas (3 clones each) and 15 clones representing five third trimester (3N, right panel) placentas (3 clones each) were sequenced. The solid and open circles represent the methylated CpG dinucleotides and unmethylated CpG, respectively. Methylation levels of each CpG sites were illustrated at the bottom panel. The CpG sites of LINE-1 in 3N placentas are generally hypomethylated relative to 1N placentas.
Figure 4.
5-Methylcytidine (5-mC) immunostaining of placental samples.
Tissue sections from paraffin-embedded placental tissues were treated with primary (Mouse anti 5-mC, concentration: 1/800), secondary (horse anti-mouse, concentration: 1/400) antibodies. Color development was performed with immunoperoxidase system. The 5-Methylcytidin-positive cells were stained brown, while negative cells displayed blue color of hematoxylin counterstaining. A. The negative control (1N) (20×10 magnitude) without primary antibody. B. 5-mC immunostaining of 1N placenta (20×10 magnitude). C. 5-mC immunostaining of 3N placenta (20×10 magnitude). Compared to 1N placenta, thinner lay of syncytium was observed in 3N placenta. Syncytiotrophoblasts (S) were stained relatively lighter than cytotrophoblast (C), stromal cells (ST), and epithelial (E) cells. V, blood vessels. Overall, similar intensity of 5-mC staining was observed 1N and 3N placentas.
Table 2.
5-mC staining scores between the groups.
Figure 5.
LINE-1 mRNA levels in first trimester (1N) and third trimester (3N) placentas and their correlation with methylation.
A. Average LINE-1 mRNA levels were 1.9 folds higher in 3N placentas compared to 1N placentas (P<0.05). B. Inverse correlation of LINE-1 mRNA levels with methylation levels in 1N and 3N placentas (Spearman correlation, rs = −0.563, P<0.05)
Figure 6.
PCNA mRNA levels in first trimester (1N) and third trimester (3N) placentas, and their association with LINE-1 mRNA levels and methylation status.
A. PCNA mRNA levels of 3N placentas were significantly higher than those of 1N placentas (4.5 folds, P<0.05). B. PCNA mRNA levels in 1N and 3N placentas were positively correlated with those of LINE-1 mRNA levels (Spearman correlation, rs = 0.702, P<0.05). C. PCNA mRNA levels in 1N and 3N placentas were negatively correlated with those of LINE-1 methylation (Spearman correlation, rs = −0.693, P<0.05).
Table 3.
Correlations between DNMTs and PCNA.
Table 4.
DNMTs mRNA levels in first trimester (1N) and third trimester (3N) placentas.