The Pcg Family Of Proteins In The Neural Tube Malformations In The Expression And Regulation

Background:Congenital defects of neural tube closure (neural tube defects, NTD) are among the commonest and most severe disorders of the fetus and newborn. Periconceptional folic acid deficiency is a risk of NTD occurrence. But there are still other unkown factors leading to NTD. Failure of the embryonic process of neural tube clousure yields anencephaly, open spinal cord and craniorachischisis. Gene-gene, gene-environment interactions are very important for the origins of NTD. Due to the affects of environmental and dietary factors, the dynamic regulation of normal development and cell growth through epigenetic mechanisms may be changed. PcGs (Polycomb group) proteins are considered as a part of core members, meanwhile microRNA (miRNA) plays a inconsiderable role in post-transcriptional gene silencing. The discovery that non-coding RNAs can repress the transcription of many genes has raised the intriguing possibility that tissue-specific expression of PcG proteins could be directed by non-coding RNA. The available knowledge in the literature about whether miRNAs anticipate in the regulatory of the PcG protein in abnormal development of neural tube is unknown. Cell apoptosis is common during embryogenesis. Teratogen can directly or indirectly disrupt the expression of key developmental genes through epigenetic mechanisms, leading to congenital defects through abnormal apoptosis. The aim of this study was to examine the regulation of PcG expression by miRNAs and the status of cell apoptosis in fetal neural tube defects induced by primary environmental risk factors.Objective:To investigate potential risk factors which can induce neural tube defect, when periconceptional folic acid is sufficient. To study regulation of PcG by miRNAs and the apoptosis during placentation and nervous system development in the fetal neural tube defects.Methods:1. ECLIA (electrochemiluminescence) is used to assay the periconceptional folic acid levels.2. The expression pattern of polycomb-group proteins in placenta and nervous tissues from NTD and normal controls was evaluated by Western blot.3. On online search of miRNA by PicTar, miRanda and TargetScan databases were used to analyze the miRNAs that regulate the expression of EED.4. TaqMan miRNA RT-Real Time PCR was used to analyse the expression of miR-30b, miR-30c, miR-181b in placenta, cerebral cortex and spinal cord.5. The identification of EED of miRNAs was studied by dual-luciferase activity assay and immunoblot analysis.6. Terminal transferase-mediated nick end labeling (TUNEL) was used to examine the apoptosis of cerebral cortex, thoracic spinal cord, and placental villi taken from fetal neural tube defects and normal controls.Results:1. The serum folic acid level in these pregnant women with NTD and normal fetus was analyzed by electrochemiluminescence. There was not significant difference between the NTDs group and normal group (P>0.05)2. EZH2, EED, BMI1, SUZ12 can be detected in variable degree in planceta and nervous tissues. EED was up-expressed in the placenta and spianl cord (P<0.05), down-expressed in the cerebral cortex (P<0.05) from NTD compared to the tissues from normal abortus.3. Bioinformations prediction showed that miR-30b, miR-30c, mi-R181b may regulate Eed expression.4. There is a converse relationship between miRNAs and its target gene. In order to analyze which miRNAs may regulation the expression of EED, the expression of miR-30b, miR-30c, miR-181b in placenta, cerebral cortex and spinal cord was detected by TaqMan miRNA RT-Real Time PCR. The expression trend of miR-30b in nervous tissues, the expression trend of miR-181b in the spinal cord and the expression trend of miR-30c in the placenta were opposite with that of EED protein level, sugestting that miR-30b, miR-30c or miR-181b possibly regulate EED expression.5. Dual-luciferase Activity Assay showed that the luciferase activity of co-transfection of miR-30b and miR-30c with PGL3-EED 3'-UTR was the lowest. It suggests that miR-30b and miR-30c can interact with 3'-untranslated region (UTR) of Eed.6. U343 cells were transfected with miR-30b or miR-30c mimics to see whether miR-30b or miR-30c up-regulation could affect endogenous EED. Compared with control, EED protein level was significantly changed when miR-30b or miR-30c was up-expressed of down-expressed (P<0.05),without any changes of Eed mRNA level.7. We detect the apoptosis in the tissuer from NTD and normal controls. The apoptotic index of exposed spinal cord was higher than non-exposed spinal cord from the spinal bifida child(P<0.05).There is no statistical difference of the apoptosis occurred in the thoracic spinal cord between NTD fetus and normal fetus(P>0.05).Excessive apoptosis was observed in the placental villi of neural tube defects compared with the normal fetus(P<0.01). 1. Under the circumstance of periconceptinal folic acid sufficiedncy, the expression of polycomb group protein has tissue-specific, and EED protein expressed statistically differently in placenta and nervous tissues from NTD.2. Under the circumstance of periconceptinal folic acid sufficiedncy, miR-30b is differently expressed in nervous tissues, while miR-30c is differently expressed in placenta tissue.3. It is the first time to identify EED is a target gene of miR-30b, miR-30c. MiR-30b, miR-30c can regulate endogenic EED's expression.4. Under the circumstance of periconceptinal folic acid sufficiedncy, aberrant cell apoptosis in placenta and nervous tissues may be one of the important ways by which human neural tube defect occurred.