Objective
The objective of the study was to investigate the expression and regulation of polycomb group (PcG) proteins in human neural tube defects (NTDs).
Study Design
PcG proteins in human NTD fetuses and age-matched controls were detected by Western blot. The relation between PcG proteins and microribonucleic acids was predicted and confirmed by the bioinformatics method, real-time polymerase chain reaction (PCR), dual-luciferase activity assay, and Western blot. The trimethyl condition of histone H3 Lys27 (H3K27) was detected by immunohistochemical and immunofluorescence.
Results
Embryonic ectoderm development protein (EED) was differentially detected in placenta, cerebral cortex, and spinal cord from NTDs and age-matched controls. MiR-30b can interact with 3′-untranslated region (UTR) of Eed and regulate endogenous EED expression in neural tissues. In addition, we found an inverse relationship between the miR-30b expression and the amount of trimethyl H3K27.
Conclusion
Differential expression of EED exists in the nerves system in human NTDs and that is regulated by miR-30b.
Neural tube defects (NTDs) are congenital malformations of the brain and spinal cord caused by neural tube close failure. NTDs occur in approximately 1 of every 1000 pregnancies in the United States and in an estimated 300,000 fetuses worldwide. In the northern provinces of China, the incidence rate of NTDs is among the highest in the world, at about 6 per 1,000 births in rural areas.
The etiology of NTDs is complicated, with both environmental and genetic contribution. Polycomb group (PcG) proteins are transcription regulatory proteins that control the expression of various genes from early embryogenesis to fetus. PcG proteins formed at least 2 classes of complexes named polycomb repressive complexes 1 (PRC1) and 2 (PRC2). PRC1 contains polycomb ring finger oncogene (BMI1), ring finger protein1, etc. PRC2 contains embryonic ectoderm development protein (EED), enhancer of zeste homolog 2 (EZH2), suppressor of zeste 12 homolog (SUZ12), etc.
Among many transcription factor genes that give rise to NTDs, PcG proteins have been found to be essential for neural tube closure. Mutations in genes that encoding most PRC1 proteins cause transformation of the axial skeleton as well as producing various neurological abnormalities by regulating Hox gene expression. Mutations in PRC2 coding genes, each of which eliminates detectable trimethyl histone H3 Lys27 (H3K27me3), cause early embryonic lethality soon after gastrulation. Although it is well known that PcG proteins participate in neurological abnormalities by regulating the downstream pathways, how the PcG proteins are regulated in occurrence of NTDs remains to be determined.
Microribonucleic acids (miRNAs), 21-24 nucleotide duplex RNAs, attenuate gene expression by pairing to 3′-untranslated region (UTR) of target transcripts inducing ribonucleic acid (RNA) cleavage or translational inhibition. The miRNAs can influence neural progenitor’s proliferation by mediating repression of target genes. The discovery that noncoding RNAs repress the transcription of many genes has raised the intriguing possibility that tissue-specific expression of PcG proteins could be regulated by noncoding RNAs. Up to now, the available knowledge in the literature about whether miRNAs anticipate in the regulatory of the PcG proteins is mainly from the report by Juan et al that miR-214 negatively feeds back on PcG by targeting the Ezh2 3′-UTR.
To explore the expression and regulation of PcG protein in abnormal development of the neural tube, we detected the expression pattern of PcG from NTDs and age-matched controls and analyzed the relation of miRNAs and PcG proteins. In this study, we found that a significant change occurred in EED expression in human NTDs. The differential expression of EED was primarily regulated by miR-30b and then changed the H3K27me3 level in nervous tissues.
Materials and Methods
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.
| Characteristic | Case | Control | P value |
|---|---|---|---|
| n | 25 | 25 | — |
| Age, y | 28.43 ± 6.77 | 29.41 ± 5.23 | .50 |
| Husband’s age, y | 30.50 ± 6.64 | 31.48 ± 5.18 | .52 |
| Nationality (Han) | 24 | 24 | 1.00 |
| Menarche age, y | 14.31 ± 1.03 | 14.53 ± 1.04 | .48 |
| Blood type (A/B/AB/O) | 8/7/1/9 | 6/7/3/9 | .76 |
| Weight, kg | 61.92 ± 8.75 | 65.50 ± 10.15 | .13 |
| Gestational week | 23.17 ± 8.28 | 25.29 ± 7.56 | .43 |
| The number of abortions | 1.27 ± 0.65 | 1.25 ± 0.46 | .88 |
| The number of spontaneous abortions | 0.21 ± 0.52 | 0.032 ± 0.18 | .093 |
| Fetal sex (female/male) | 19/6 | 20/5 | .88 |
| Member with regulatory menstrual cycle | 21 | 24 | .22 |
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.
Results
The expression pattern of PcG proteins in placenta, cerebral cortex, and spinal cord
The expression pattern of different PcG proteins in the different tissues from NTDs and normal controls was diversity ( Figure 1 ). In the placenta, the expression level of EZH2 and BMI1 was not recognizably different between NTDs and the control group. SUZ12 protein expressed more in NTDs than that in the controls ( P < .05). The protein level of the EED was higher in NTD fetuses than that in normal fetuses ( P < .05).
In the cerebral cortex, the expression of EZH2, BMI1, and SUZ12 was not significantly different between the case and control groups. The EED protein level was significantly decreased in the NTDs compared with the controls ( P < .05).
In the spinal cord, the expression of BMI1 and SUZ12 was hardly detected in all the samples. The EZH2 protein level was higher in NTDs than that in controls ( P < .05). The expression of EED was stronger in the NTDs than that in the normal fetuses ( P < .05). These results showed that EED was detected differentially in all tissues, particularly in nervous tissues.
Prediction of miRNAs that regulate EED expression
To explore whether miRNAs anticipate in the regulation of EED expression, an online search of miRNA by miRanda ( http://cbio.mskcc.org/cgi-bin/mirnaviewer/mirnaviewer.pl ), and TargetScan databases ( http:genes.mit.edu/targetscan.test/ucsc.html ) were used to analyze the miRNAs that regulated EED expression and found that EED expression may be modulated by miR-30b , miR-30c , and miR-181b ( Figure 2 , A). As shown in Figure 2 , Aa, both miR-30b and miR-30c bind to the same 2 miRNA responsive elements (MREs), and miR-181b binds to a single MRE in the 3′-UTR of Eed .
