Sample collections

A population-based embryo development defects survey was performed in the Family Planning Technique Service Station in Qian’xi, Hebei Province, from December 2006 to December 2008.

Fifty mothers containing harbored NTDs fetuses and age-matched controls were surveyed by a questionnaire that contained the general condition ( Table ). The serum folic acid level in these pregnant women was analyzed by electrochemiluminescence ( Supplemental Table ). There was no significant difference between the NTD group and the normal group. Twenty-five fetuses with NTDs (anencephaly, n = 11; spina bifida, n = 14) were taken as subjects and age-matched normal fetuses as controls.

The control groups were obtained from allowable therapeutic abortions. In the control group, therapeutic abortions were performed when there was a serious threat to the mother’s health or for a lethal threat, for example, the accident or serious pregnancy reaction to pregnant mother. In the case group, induced abortions were performed when the fetus was found to have spina bifida or anencephaly by B-mode ultrasound. The placenta, cerebral cortex, and spinal cord were excised and frozen in liquid nitrogen for RNA and protein analysis.

This study was approved by the Ethics Committee of the National Research Institute for Family Planning. The collection of fetal tissues followed the procedures that are in accordance with the ethical standards as formulated in the Helsinki Declaration.

RNA and protein extraction

To detect the expression of PcG proteins and miRNAs in the placenta, spinal cord, and cerebral cortex, we extracted protein and total RNA from these tissues. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s standard protocol and total protein using Cellytic mammalian tissue lysis/extraction reagent (Sigma, St Louis, MO).

Western blot analysis

Western blot analysis was used to detect the expression of PcG proteins. Seventy-five microgram proteins were separated by electrophoresis, and the proteins in the gels were blotted onto polyvinylidene fluoride membranes (Amersham, St Albans, Herts, UK) by electrophoretic transfer. The membrane was incubated with rabbit anti-EZH2, BMI1, EED, or SUZ12 polyclonal antibody or mouse anti-β-actin monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. The specific protein-antibody complexes were detected by using horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Detection by the chemiluminescence reaction was carried using the enhanced luminol-based chemiluminescent kit (Millipore, Billerica, MA). The β-actin signal was used as a loading control. The experiment has been repeated at least 3 times. The bands were analyzed using Quantity One analyzing system (Bio-Rad Laboratories, Hercules, CA).

Cell cultures and transfection

Cell cultures and transfection were used to detect the relations of EED and miRNAs and the effect of miRNAs on endogenous EED expression and H3K27me3 level. U343 cells were cultured in Dulbecco’s modified Eagle’s medium/nutrient F-12 Ham’s (DMEM/F-12) culture medium (Hyclone, Logan, UT), supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 10 mg/ml streptomycin.

The miRNA mimics, miRNA inhibitor, pre-miR control, or anti-miR control purchased from GenePharma Co (Shanghai, China) was transfected into the U343 cells by using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. Forty-eight hours later, the cells were collected to extract protein and RNA.

Real-time reverse transcript polymerase chain reaction (RT-PCR)

Real time RT- PCR was used to detect the expression of EED. Total RNA (2 μg) was used as template for reverse transcription using Superscript III (Invitrogen). The mRNA levels of Eed and glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) were measured by real-time PCR using a FastStart universal SYBR Green master (Roche, Mannheim, Germany) and ABI prism7000 sequence detection system (Applied Biosystems, Foster City, CA). The sequences of primers were as follows: For Eed , forward, 5′-AGAAGCTGAGCAGTGACGAGAACA-3′ and reverse 5′-AGGTGCATTTGGCG TGTTTGTAGG-3′; and for Gapdh , forward 5′-TGATGTGTTCCAACCAGAGGAT-3′ and reverse 5′-GGTGAGAGGGAAGAGCTGAAGT-3′. Each sample in each group was measured in triplicate and the experiment was repeated at least 3 times. The quantification was normalized to an endogenous control Gapdh.

TaqMan miRNA assay

TaqMan miRNA assay was used to detect the expression of miRNAs that regulated EED. Single-stranded cDNA was synthesized by using TaqMan miRNA reverse transcription kit (Applied Biosystems) and then amplified by using TaqMan universal PCR master mix (Applied Biosystems) together with miRNA-specific TaqMan MGB probes: miR-30b , miR-30c , or miR-181b (Applied Biosystems). Each sample in each group was measured in triplicate and the experiment was repeated at least 3 times. The U6 small nuclear RNA was used for normalization.

Luciferase (LUC) activity assay

Luciferase activity assay was used to detect the interaction between 3′-UTR of Eed and miRNAs. A 166 nt long region of the 3′-UTR of Eed was amplified from human genomic DNA and cloned into the downstream of the stop site of luciferase coding gene in pGL3. Eed 3′-UTR, deleted miR-30b , miR-30c , and miR-181b target sites, was used as control vector ( Supplemental Figure S1 ). For the luciferase assay, U343 cells were seeded into 96 well plates. The cells were cotransfected with Eed 3′-UTR inserted vector or control vector, renilla luciferase expression vector, and sythesized miRNA mimics, inhibitors, and controls. Two days later, cells were harvested and assayed using the dual-luciferase assay kit (Promega, Madison, WI). Each treatment was performed triplicate in 3 independent experiments. The results were expressed as relative luciferase activity (firefly LUC/Renilla LUC).

Immunohistochemistry

Immunohistochemistry was used to analyze the H3K27me3 level in tissues. Paraffin-embedded microarray slides (5 μm) including placenta, cerebral cortex, and spinal cord were deparaffinized and rehydrated and then incubated with anti-H3K27 polyclonal antibody (Millipore) and corresponding HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories). Positive cells were counted in different optical fields (magnification ×400) selected in a random manner and counted at least 500 cells for each sample.

Immunofluorescence

Immunofluorescence was used to analyze the H3K27me3 level in cells cultured in vitro. Adherent cells cultured on chamber slides were fixed with 4% paraformaldehyde in phosphate-buffered saline and then incubated with anti-H3K27 polyclonal antibody (Millipore) and corresponding fluorescein isothiocyanate-conjugated secondary antibody (Jackson ImmunoResearch Laboratories). Cells were counterstained with Hoechst 33342 (10 μg/mL). Cells were examined with a Zeiss LSM5 PASCAL microscope (Jena, Germany). Positive cells were counted in different optical fields (magnification ×400) selected in a random manner and counted at least 500 cells for each sample.

Statistical analysis

Western blot results of NTDs and normal controls were estimated using the rank sum test to analyze the association between the PcG expression and the NTDs’ occurrence. P < .05 was considered significant.

The results of Western blot, real-time PCR, immunohistochemistry, and immunofluorescence were analyzed by 1 way analysis of variance. All values are reported as the mean ± SE. When significant effects of treatment were indicated, Duncan’s multiple-range test was used for group comparisons. All statistical analyses were performed using SPSS version 14.0 (SPSS Inc, Chicago, IL). A value of P < .05 was considered statistically significant.