Role of microRNA-132 and Mecp2 in methamphetamine dependence / 中国药理学通报

Aim To explore the roles of miRNA-132 and its related proteins(Mecp2, CREB)in the mechanism of methamphetamine(MA)-induced neurotoxicity and dependence.Methods The rats were intraperitioneally injected(ip)with MA(10 mg·kg-1·d-1)to establish methamphetamine dependence model with different dependent time courses of 1 week, 2 weeks, and 4 weeks respectively.The miRNA-132 and Mecp2 mRNA were detected by RT-qPCR, and the Mecp2, p-Mecp2, CREB and p-CREB proteins were detected by Western blot in the tissues of frontal cortex and hippocampus.Results In the frontal cortex, the miRNA-132 and Mecp2 mRNA were up-regulated in MA-dependent groups(P<0.05 and P<0.01), while the Mecp2 protein were down-regulated(P<0.01).MA could promote the phosphorylation of Mecp2 protein in the frontal cortex(P<0.01).In hippocampus, the miRNA-132 was down-regulated in the MA-dependent groups, but Mecp2 mRNA was up-regulated(P<0.05).Mecp2 protein increased in MA-dependent 1 week group(P<0.05), and then recovered with the prolonged time of MA dependence, then decreased in MA-dependent 4 weeks groups(P<0.05)in hippocampus.The phosphorylation level of Mecp2 was significantly decreased in the 1 week group(P<0.01), and then increased in the 2 weeks group(P<0.01)in hippocampus.Conclusions MA could induce an abnormal expression of miRNA-132 in the frontal cortex and hippocampus, and miRNA-132 might inhibit the translation of Mecp2 mRNA and induce the decrease expression of Mecp2 protein in the frontal cortex.But in hippocampus, miRNA-132 does not show the correlation with the Mecp2 expression trend of the frontal cortex.And miRNA-132 regulation does not depend on the expression of Mecp2 in hippocampus.

The Role of MeCP2 in Substance Addiction / 中国生物化学与分子生物学报

Substance addiction is considered to be a chronic recurrent encephalopathy. The neural adap-tation changes induced by addictive substances are partly mediated by epigenetic mechanism. The perma￣nent changes of gene expression in tissues or brain can be affected by DNA methylation, histone modifica-tion and chromatin remodeling, these changes eventually lead to behavioral abnormalities of individual. Methyl-CpG binding protein 2 (MeCP2), an important transcription inhibitor, contains characteristic do-mains that regulated chromosome conformation, transcription and RNA splicing. It has also been identified that MeCP2 plays an important role in regulating neuronal plasticity and related target gene transcription during brain development, which add more attention about the importance of epigenetic mechanism in neuronal function. Studies have showed that DNA methylation, histone acetylation and phosphorylation regulate MeCP2 gene expression, affect gene and protein transcription, translation and cell regulation in learning, memory and substance addiction. Addictive substances induce psychological and mental dependence, which are related to the changes of neuronal plasticity and gene expression in addictive neural circuits. MeCP2 plays an important role in regulating synaptic transmission and neuronal plasticity in central nervous system. Therefore, it is of great scientific significance to explore the role of MeCP2 in regulating neuronal plasticity in the central nervous system. In this review we summarized the structure and function of MeCP2, the relationship between MeCP2 and epigenetics, and the role of MeCP2 epigenetic modifications induced by different addictive substances in substance addiction, which may provide further understanding of the molecular mechanism of substance addiction and provide new in￣sight for clinical intervention.

Regulation and its mechanism of MeCP2 on biological behavior and epithelial-mesenchymal transition of LECs / 中华实验眼科杂志

Objective@#To investigate the role of methyl-CpG-binding protein 2 (MeCP2) in the regulation and epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs) and its possible mechanism.@*Methods@#Human LEC lines (SRA01/04) were divided into MeCP2-mimic group, MeCP2-NC group and small interferening RNA-MeCP2 (si-MeCP2) group, and MeCP2 analog plasmid, blank plasmid and MeCP2 si-RNA plasmid was used respectively to transfect the cells.The expression of MeCP2 mRNA in the cells was detected by using real-time PCR 24 hours after transfection.At 48 hours after transfection, the migration rate of the cells was evaluated by scratching test, and the expression of Wnt3a protein in the cells was detected by immunofluorescence stainning.The relative expressions of β-catenin, E-cadherin, Vimentin, matrix metallo proteinase (MMP)-9, MMP-7 and secreted frizzled-related protein 5 (SFRP5) proteins in the cells were detected by Western blot.@*Results@#After 24 hours of transfection, the relative expression of MeCP2 mRNA in the cells was significantly different among the MeCP2-mimic group, MeCP2-NC group and si-MeCP2 group (F=4 773.00, P<0.00 1). The migrating rate of the cells in the MeCP2-mimic group, MeCP2-NC group and si-MeCP2 group was (57.45±5.20)%, (32.71±10.02)% and (17.77±9.22)%, respectively, showing a significant difference among the three groups (F=124.00, P<0.001), and the migrating rate of the cells in the si-MeCP2 group was significantly lower than that of the MeCP2-mimic group or MeCP2-NC group (both at P<0.001). The relative expressing intensity (absorbance) of Wnt3a in the cells of the MeCP2-mimic group, MeCP2-NC group and si-MeCP2 group was 75.92±6.10, 52.03±5.22 and 28.75±3.39, respectively, with a significant difference among three the groups (F=221.30, P<0.001), and the relative expressing intensity (absorbance) of Wnt3a in the cells was significantly lower in the si-MeCP2-mimic group than that of the MeCP2-NC group and MeCP2-mimic group (both at P<0.001). The relative expressing level of E-cadherin protein was significantly elevated and the expressions of β-catenin, Vimentin, MMP-9 and MMP-7 were significantly reduced in the si-MeCP2 group compared with the MeCP2-mimnic group and MeCP2-NC group (all at P<0.01). The relative expressing level of SFRP5 protein in the MeCP2-mimic group, MeCP2-NC group and si-MeCP2 group was 27.19±0.03, 47.54±0.05 and 74.93±0.05, respectively, showing a statistical difference among the three groups (F=183.49, P<0.001), and the relative expressing level of SFRP5 in the si-MeCP2 group was significantly higher than that in the MeCP2-mimic group and MeCP2-NC group (both at P<0.001).@*Conclusions@#MeCP2C can promote EMT of human LECs by down-regulating the expression of SFRP5 and therefore activating the Wnt3a/β-catenin signal pathway.

Regulation and its mechanism of MeCP2 on biological behavior and epithelial-mesenchymal transition of LECs / 中华实验眼科杂志

Objective To investigate the role of methyl-CpG-binding protein 2 (MeCP2) in the regulation and epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs) and its possible mechanism.Methods Human LEC lines (SRA01/04) were divided into MeCP2-mimic group,MeCP2-NC group and small interferening RNA-MeCP2 (si-MeCP2) group,and MeCP2 analog plasmid,blank plasmid and MeCP2 si-RNA plasmid was used respectively to transfect the cells.The expression of MeCP2 mRNA in the cells was detected by using real-time PCR 24 hours after transfection.At 48 hours after transfection,the migration rate of the cells was evaluated by scratching test,and the expression of Wnt3a protein in the cells was detected by immunofluorescence stainning.The relative expressions of β-catenin,E-cadherin,Vimentin,matrix metallo proteinase (MMP)-9,MMP-7 and secreted frizzled-related protein 5 (SFRP5) proteins in the cells were detected by Western blot.Results After 24 hours of transfection,the relative expression of MeCP2 mRNA in the cells was significantly different among the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group (F =4 773.00,P＜0.00 1).The migrating rate of the cells in the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group was (57.45±5.20)％,(32.71± 10.02)％ and (17.77±9.22)％,respectively,showing a significant difference among the three groups (F=124.00,P＜0.001),and the migrating rate of the cells in the si-MeCP2 group was significantly lower than that of the MeCP2-mimic group or MeCP2-NC group (both at P＜0.001).The relative expressing intensity (absorbance) of Wnt3a in the cells of the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group was 75.92 ± 6.10,52.03 ± 5.22 and 28.75 ± 3.39,respectively,with a significant difference among three the groups (F=221.30,P＜0.001),and the relative expressing intensity (absorbance) of Wnt3a in the cells was significantly lower in the si-MeCP2-mimic group than that of the MeCP2-NC group and MeCP2-mimic group (both at P＜0.001).The relative expressing level of E-cadherin protein was significantly elevated and the expressions of β-catenin,Vimentin,MMP-9 and MMP-7 were significantly reduced in the si-MeCP2 group compared with the MeCP2-mimnic group and MeCP2-NC group (all at P＜O.01).The relative expressing level of SFRP5 protein in the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group was 27.19± 0.03,47.54±0.05 and 74.93±0.05,respectively,showing a statistical difference among the three groups (F =183.49,P＜0.001),and the relative expressing level of SFRP5 in the si-MeCP2 group was significantly higher than that in the MeCP2-mimic group and MeCP2-NC group (both at P＜0.001).Conclusions MeCP2C can promote EMT of human LECs by down-regulating the expression of SFRP5 and therefore activating the Wnt3a/β-catenin signal pathway.

Síndrome de Rett: Análisis molecular del gen MECP2 en pacientes chilenas / Rett syndrome: MECP2 gene molecular analysis in Chilean patients

INTRODUCCIÓN: El síndrome de Rett (RTT) es un trastorno neurológico progresivo caracterizado por producir una regresión del desarrollo psicomotor en niñas previamente sanas. La mayoría de los casos son causados por variantes patogénicas en el gen MECP2, que codifica para la proteína methyl CpG- binding protein 2. OBJETIVO: Describir la frecuencia y el tipo de variantes patogénicas en MECP2 en mujeres chilenas con diagnóstico clínico de RTT. PACIENTES Y MÉTODO: Se invitó a participar en este estudio a mujeres chilenas con sospecha clínica de RTT. Se reunió información clínica mediante un cuestionario. Se analizaron variantes patogénicas en MECP2 mediante el método de secuenciación de Sanger y se utilizó Multiple Ligation-dependant Probe Amplification (MLPA) para la detección de duplicaciones y deleciones. RESULTADO: El estudio incluyó 14 pacientes con sospecha de RTT, de las cuales 8 (57%) pacientes tuvieron variantes patogénicas. Las restantes permanecen sin diagnóstico molecular. CONCLUSIÓN: Variantes patogénicas en MECP2 están presentes en pacientes chilenas con RTT. Es probable que haya otros genes o diagnósticos involucrados en las pacientes sin hallazgos en MECP2. A partir de este trabajo, el diagnóstico molecular está disponible en Chile.

INTRODUCTION: Rett syndrome (RTT) is a progressive neurological disorder characterized by regres sion of psychomotor development in previously healthy girls. Most cases are due to pathogenic va riants in the MECP2 gene which encodes for the methyl CpG-binding protein 2. OBJECTIVE: To des cribe the frequency and type of pathogenic variants in the MECP2 gene in Chilean female patients with clinical diagnosis of RTT. PATIENTS AND METHOD: Chilean women with clinical suspicion of RTT were invited to participate in the study. Clinical data were collected through a questionnaire. MECP2 pathogenic variants were analyzed by Sanger sequencing method and Multiplex Ligation-dependent Probe Amplification (MLPA) was used to detect duplications or deletions. RESULTS: The study in cluded 14 patients with suspected RTT, of which eight (57%) patients had pathogenic variants. The other patients remain without molecular diagnosis. CONCLUSIONS: Pathogenic variants in MECP2 are present in Chilean patients with RTT. It is likely that there are other genes or diagnoses involved in patients without MECP2 findings. As of this study, molecular diagnosis is available in Chile.

The different expression and significance of methyl CpG binding protein 2 and histone deacetylase 6 in human scar formation / 中华整形外科杂志

Objective@#To investigate the expression and significance of fibroblasts methyl CpG binding protein 2(MECP2)and histone deacetylase 6(HDAC6) at different stages of scar tissues and hypertrophic scars.@*Methods@#From August 2016 to May 2017, 118 cases of scar and 33 cases of upper eyelid lasia received from the Department of Plastic and Burn Surgery of the First Affiliated Hospital of Chongqing Medical University, Chongqing were collected, including 33 cases of normal skin (ages 34-68, 12 cases of male and 21 cases of female), 32 cases of normal scar (ages 8-68, 19 cases of male and 13 cases of female), 35 cases of keloid (ages 11-62, 11 cases of male and 24 cases of female) and 51 cases of hypertrophic scar (ages 12-58, 22 cases of male and 29 cases of female) without any treatment.The hypertrophic scar was divided into 4 groups according to the growth time: 10 cases in the 0-3 month group, 11 cases in the 4-6 month group, 13 cases in the 7-12 month group, and 17 cases in the >12 month group. Immunohistochemistry and western blot were used to detect the expressions of MECP2 and HDAC6 in tissues of each group, and real-time quantitative polymerase chain reaction gene amplification fluorescence detection technology was used to detect the expressions of MECP2mRNA and HDAC6 mRNA, and the differences between each group were compared. SPSS20.0 was used for statistical analysis of the data obtained in the experiment, and one-way ANOVAwas used to test the differences between the groups. P<0.05 was considered statistically significant.@*Results@#MECP2 protein is gradually expressed in normal skin (1 326.4±572.3), normal scar (2 341.4±816.2), hypertrophic scar (3 500.7±1407.6) and keloid (4 787.9±1 514.3), F=33.82, P=0.001. Similarly, the expression of MECP2mRNA in normal skin (0.82±0.43), normal scar (1v14±0.45), hypertrophic scar (1.59±0.39) and keloid (2.14±0.53) was also gradually increased, F=23.4, P=0.001. In normal skin (1 332.5±746.7) and normal scar (2 307.7±1 027.4), hypertrophic scar (4 107.4±1 223.1) and keloid, the level of HDAC6 protein expression (5 155.9±1 265.3) were significant different, F = 50.27, P=0.001.There were still statistically significant differences in HDAC6mRNA between normal skin (0.57±0.23), normal scar(1.03±0.35), hypertrophic scar (1.47±0.31) and keloid (1.87±0.45), F=38.06, P=0.001.In hypertrophic scar, there was no significant difference in MECP2 between the 0-3 month group (4 758.4±660.1) and the 4-6 month group (4 602.4 ±583.9), F=1.28, P=0.97), 7-12 month (3 000.7±982.7) group and >12 month (1 990.7 ±992.3)group of MECP2 expression quantity decrease, F=25.8, P=0.001; There was no significant difference in the level of HDAC6 protein in the 0-3 month (5 069.3±1 236.1) group and 4-6 month (5 316.7±1 237.4) group, F=1.02, P=0.98, and the expression was significantly reduced in the 7-12 month (3 084.7±1 685.5) group and the>12 month (2 304.6±1 337.1)group, F=11.41, P=0.001.@*Conclusions@#MECP2 and HDAC6 play a critical role in the formation of scarring in the human body. Epigenetic regulation mediated by MECP2 and HDAC6 is an important target for inhibiting scar formation.

Dual mechanisms for the regulation of brain-derived neurotrophic factor by valproic acid in neural progenitor cells

Autism spectrum disorders (ASDs) are neurodevelopmental disorders that share behavioral features, the results of numerous studies have suggested that the underlying causes of ASDs are multifactorial. Behavioral and/or neurobiological analyses of ASDs have been performed extensively using a valid model of prenatal exposure to valproic acid (VPA). Abnormal synapse formation resulting from altered neurite outgrowth in neural progenitor cells (NPCs) during embryonic brain development has been observed in both the VPA model and ASD subjects. Although several mechanisms have been suggested, the actual mechanism underlying enhanced neurite outgrowth remains unclear. In this study, we found that VPA enhanced the expression of brain-derived neurotrophic factor (BDNF), particularly mature BDNF (mBDNF), through dual mechanisms. VPA increased the mRNA and protein expression of BDNF by suppressing the nuclear expression of methyl-CpG-binding protein 2 (MeCP2), which is a transcriptional repressor of BDNF. In addition, VPA promoted the expression and activity of the tissue plasminogen activator (tPA), which induces BDNF maturation through proteolytic cleavage. Trichostatin A and sodium butyrate also enhanced tPA activity, but tPA activity was not induced by valpromide, which is a VPA analog that does not induce histone acetylation, indicating that histone acetylation activity was required for tPA regulation. VPA-mediated regulation of BDNF, MeCP2, and tPA was not observed in astrocytes or neurons. Therefore, these results suggested that VPA-induced mBDNF upregulation was associated with the dysregulation of MeCP2 and tPA in developing cortical NPCs.

Interaction between abnormal expression of fragile histidine triad and methyl-CpG-binding protein 2 on cervical cancerization / 中华流行病学杂志

Objective To explore the relationship between abnormal expression of fragile histidine triad (FHIT) gene and methyl-CpG-binding protein 2 (MeCP2) as well as their interaction on cervical cancerization.Methods A total of 73 patients with cervical squamous cell carcinoma (SCC),113 patients with cervical intraepithelial neoplasia (CIN Ⅰ,n =45;CIN Ⅱ/Ⅲ,n=68) and 60 women with normal cervix (NC) were included in the study.Real time PCR and Western blot were performed to detect the expression levels of mRNA and protein about FHIT and MeCP2,respectively.The methylation status of FHIT gene CpG island was tested by methylation-specifc PCR (MSP).Kruskal-Wallis H test,x2 test,trend x2 test and Spearman correlation analysis were conducted with software SPSS 20.0.The interaction was evaluated by generalized multifactor dimensionality reduction (GMDR) model.Results With the deterioration of cervical lesion,the methylation rates of FHIT gene CpG island (x2=18.64,P＜0.001;trendx2=18.08,P＜0.001) increased gradually,while the expression levels of FHIT mRNA (H=27.32,P＜0.001;trendx2=12.65,P＜0.001) and protein (H=47.10,P＜0.001;trendx2=29.79,P＜0.001) decreased gradually.There was a negative correlation between the methylation rates of FHIT gene CpG island and the expression level of FHIT protein (r=-0.226,P＜0.001).The levels of MeCP2 mRNA (H=26.19,P＜0.001;trend x2=11.81,P=0.001) and protein (H=69.02,P＜ 0.001;trend x2 =47.44,P＜ 0.001) increased gradually with the aggravation of cervical lesions.There was a positive correlation between the expression level of MeCP2 protein and the FHIT mRNA Ct ratio (r=0.254,P＜0.001).Expression of proteins were negatively correlated between MeCP2 and FHIT (r=-0.213,P=0.001).The results analyzed by GMDR model showed that there were interactions among high MeCP2 protein expression,the CpG island methylation of FHIT and mRNA and protein expression in CIN Ⅱ/Ⅲ group,and among high MeCP2 mRNA and protein expression,the CpG island methylation of FHIT and low mRNA and protein expression in SCC group.Conclusion High expression of MeCP2 mRNA and protein,the CpG island methylation and low mRNA and protein expression of FHIT could increase the risk of cervical carcinogenesis,and there might be a synergistic effect on cervical carcinogenesis.

Interaction between abnormal expression of fragile histidine triad and methyl-CpG-binding protein 2 on cervical cancerization / 中华流行病学杂志

Objective To explore the relationship between abnormal expression of fragile histidine triad (FHIT) gene and methyl-CpG-binding protein 2 (MeCP2) as well as their interaction on cervical cancerization.Methods A total of 73 patients with cervical squamous cell carcinoma (SCC),113 patients with cervical intraepithelial neoplasia (CIN Ⅰ,n =45;CIN Ⅱ/Ⅲ,n=68) and 60 women with normal cervix (NC) were included in the study.Real time PCR and Western blot were performed to detect the expression levels of mRNA and protein about FHIT and MeCP2,respectively.The methylation status of FHIT gene CpG island was tested by methylation-specifc PCR (MSP).Kruskal-Wallis H test,x2 test,trend x2 test and Spearman correlation analysis were conducted with software SPSS 20.0.The interaction was evaluated by generalized multifactor dimensionality reduction (GMDR) model.Results With the deterioration of cervical lesion,the methylation rates of FHIT gene CpG island (x2=18.64,P＜0.001;trendx2=18.08,P＜0.001) increased gradually,while the expression levels of FHIT mRNA (H=27.32,P＜0.001;trendx2=12.65,P＜0.001) and protein (H=47.10,P＜0.001;trendx2=29.79,P＜0.001) decreased gradually.There was a negative correlation between the methylation rates of FHIT gene CpG island and the expression level of FHIT protein (r=-0.226,P＜0.001).The levels of MeCP2 mRNA (H=26.19,P＜0.001;trend x2=11.81,P=0.001) and protein (H=69.02,P＜ 0.001;trend x2 =47.44,P＜ 0.001) increased gradually with the aggravation of cervical lesions.There was a positive correlation between the expression level of MeCP2 protein and the FHIT mRNA Ct ratio (r=0.254,P＜0.001).Expression of proteins were negatively correlated between MeCP2 and FHIT (r=-0.213,P=0.001).The results analyzed by GMDR model showed that there were interactions among high MeCP2 protein expression,the CpG island methylation of FHIT and mRNA and protein expression in CIN Ⅱ/Ⅲ group,and among high MeCP2 mRNA and protein expression,the CpG island methylation of FHIT and low mRNA and protein expression in SCC group.Conclusion High expression of MeCP2 mRNA and protein,the CpG island methylation and low mRNA and protein expression of FHIT could increase the risk of cervical carcinogenesis,and there might be a synergistic effect on cervical carcinogenesis.

Puerarin Up-regulates Methyl-CpG Binding Protein 2 Phosphorylation in Hippocampus of Vascular Dementia Rats / 中国结合医学杂志

<p><b>OBJECTIVE</b>To observe the effect of puerarin on methyl-CpG binding protein 2 (MeCP2) phosphorylation (pMeCP2) in the hippocampus of a rat model of vascular dementia (VD).</p><p><b>METHODS</b>Thirty-six healthy Sprague-Dawley rats were randomly assigned to the sham-operated group, dementia group and puerarintreated group using a random number table (n=12 per group). The modifified permanent bilateral common carotid artery occlusion method was used to establish the VD model. The sham-operated and dementia groups were given 2 mL/d of saline, while the puerarin-treated group was given 100 mg/(kg•d) of puerarin for 17 days. The learning and memory abilities were evaluated by the Morris water maze test. Hematoxylin-eosin staining, immunohistochemical (IHC) staining and Western blot analysis were carried out to observe changes in neuron morphology and in level of pMeCP2 in the hippocampus, respectively.</p><p><b>RESULTS</b>The morphologies of rat hippocampal neurons in the puerarintreated group were markedly improved compared with the dementia group. The escape latency of the dementia group was significantly longer than the sham-operated group (P<0.05), while the puerarin-treated group was obviously shorter than the dementia group (P<0.05). Cross-platform times of the dementia group were signifificantly decreased compared with the sham-operated group (P<0.05), while the puerarin-treated group was obviously increased compared with the dementia group (P<0.05). IHC staining showed no significant difference in the number of MeCP2 positive cells among 3 groups (P>0.05). The number of pMeCP2 positive cells in the CA1 region of hippocampus in the dementia group was signifificantly increased compared with the sham-operated group, and the puerarin-treated group was signifificantly increased compared with the dementia group (both P<0.05). Western blot analysis showed no signifificant difference of MeCP2 expression among 3 groups (P>0.05). The expression of pMeCP2 in the dementia group was signifificantly increased compared with the sham-operated group, while it in the puerarin-treated group was signifificantly increased compared with the dementia group (P<0.05).</p><p><b>CONCLUSION</b>Puerarin could play a role in the protection of nerve cells through up-regulating pMeCP2 in the hippocampus, improving neuron morphologies, and enhancing learning and memory ablities in a rat model of VD.</p>

Changes in expression of H3K9Ac and MeCP2 in spinal cord and dorsal root ganglion of rats with diabetic neuropathic pain / 中华麻醉学杂志

Objective To evaluate the changes in the expression of acetylated histone 3 lysine 9 (H3K9Ac) and methyl-CpG-binding protein 2 (MeCP2) in the spinal cord and dorsal root ganglion (DRG) of rats with diabetic neuropathic pain (DNP).Methods Pathogen-free male Sprague-Dawley rats,weighing 120-160 g,were fed a high-fat and high-sucrose diet for 8 weeks,then streptozocin 35 mg/kg was intraperitoneally injected,and type 2 diabetes mellitus was confirmed by blood glucose level ≥ 16.7 mmol/L 3 days later.DNP model was considered successful when the decrease in pain threshold ＜ 85％ of the baseline value on 14 days after injection,otherwise it was considered as non-DNP (NDNP).Eighteen rats with DNP and NDNP served as DNP and NDNP groups,respectively,and another 18 normal rats served as control group (group C).At 3,7 and 14 days after successful establishment of the model,the mechanical paw withdrawal threshold and thermal paw withdrawal latency were measured in group DNP and at the corresponding time points in C and NDNP groups,and then rats were sacrificed,and the lumbar segment of the spinal cord and DRGs were removed for determination of the expression of H3K9Ac and MeCP2 by Western blot.Results Compared with C and NDNP groups,mechanical paw withdrawal threshold was significantly decreased,thermal paw withdrawal latency was shortened,the expression of H3K9Ac in the spinal cord and DRGs was up-regulated,and the expression of MeCP2 in the spinal cord and DRGs was down-regulated at 3,7 and 14 days after successful establishment of the model in group DNP (P＜0.05).Conclusion The maintenance mechanism of DNP may be related to up-regulated expression of H3K9Ac and down-regulated expression of MeCP2 in rats with DNP.

Functional Profiling of Human MeCP2 by Automated Data Comparison Analysis and Computerized Expression Pathway Modeling / 대한의료정보학회지

OBJECTIVES: Methyl-CpG binding protein 2 (MeCP2) is a ubiquitous epigenetic factor that represses gene expression by modifying chromatin. Mutations in the MeCP2 gene cause Rett syndrome, a progressive neurodevelopmental disorder. Recent studies also have shown that MeCP2 plays a role in carcinogenesis. Specifically, functional ablation of MeCP2 suppresses cell growth and leads to the proliferation of cancer cells. However, MeCP2's function in adult tissues remains poorly understood. We utilized a weight matrix-based comparison software to identify transcription factor binding site (TFBS) of MeCP2-regulated genes, which were recognized by cDNA microarray analysis. METHODS: MeCP2 expression was silenced using annealed siRNA in HEK293 cells, and then a cDNA microarray analysis was performed. Functional analysis was carried out, and transcriptional levels in target genes regulated by MeCP2 were investigated. TFBS analysis was done within genes selected by the cDNA microarray analysis, using a weight matrix-based program and the TRANSFAC 6.0 database. RESULTS: Among the differentially expressed genes with a change in expression greater than two-fold, 189 genes were up-regulated and 91 genes were down-regulated. Genes related to apoptosis and cell proliferation (JUN, FOSL2, CYR61, SKIL, ATF3, BMABI, BMPR2, RERE, and FALZ) were highly up-regulated. Genes with anti-apoptotic and anti-proliferative functions (HNRPA0, HIS1, and FOXC1) were down-regulated. Using TFBS analysis within putative promoters of novel candidate target genes of MeCP2, disease-related transcription factors were identified. CONCLUSIONS: The present results provide insights into the new target genes regulated by MeCP2 under epigenetic control. This information will be valuable for further studies aimed at clarifying the pathogenesis of Rett syndrome and neoplastic diseases.

Functional Profiling of Human MeCP2 by Automated Data Comparison Analysis and Computerized Expression Pathway Modeling / 대한의료정보학회지

OBJECTIVES: Methyl-CpG binding protein 2 (MeCP2) is a ubiquitous epigenetic factor that represses gene expression by modifying chromatin. Mutations in the MeCP2 gene cause Rett syndrome, a progressive neurodevelopmental disorder. Recent studies also have shown that MeCP2 plays a role in carcinogenesis. Specifically, functional ablation of MeCP2 suppresses cell growth and leads to the proliferation of cancer cells. However, MeCP2's function in adult tissues remains poorly understood. We utilized a weight matrix-based comparison software to identify transcription factor binding site (TFBS) of MeCP2-regulated genes, which were recognized by cDNA microarray analysis. METHODS: MeCP2 expression was silenced using annealed siRNA in HEK293 cells, and then a cDNA microarray analysis was performed. Functional analysis was carried out, and transcriptional levels in target genes regulated by MeCP2 were investigated. TFBS analysis was done within genes selected by the cDNA microarray analysis, using a weight matrix-based program and the TRANSFAC 6.0 database. RESULTS: Among the differentially expressed genes with a change in expression greater than two-fold, 189 genes were up-regulated and 91 genes were down-regulated. Genes related to apoptosis and cell proliferation (JUN, FOSL2, CYR61, SKIL, ATF3, BMABI, BMPR2, RERE, and FALZ) were highly up-regulated. Genes with anti-apoptotic and anti-proliferative functions (HNRPA0, HIS1, and FOXC1) were down-regulated. Using TFBS analysis within putative promoters of novel candidate target genes of MeCP2, disease-related transcription factors were identified. CONCLUSIONS: The present results provide insights into the new target genes regulated by MeCP2 under epigenetic control. This information will be valuable for further studies aimed at clarifying the pathogenesis of Rett syndrome and neoplastic diseases.

Clinical features, gene mutation and genetic counseling in Rett syndrome patients / 中华实用儿科临床杂志

Objective To investigate the mutations in methyl-CpG-binding protein 2 gene (MECP2 gene) from typical sporadic Rett syndrome patients,explore the correlations between their genotype and phenotype,assist in genetic counseling.Methods Genomic DNA was extracted from peripheral blood leukocytes from 2 patients and their parents using standard protocols.Polymerase chain reaction and direct sequencing were performed using specific primers from 4 exons in MECP2 gene.Results No mutations were found in exon 1,2,3.Two different heterozygous missense mutations in exon 4 within MECP2 gene were identified from 2 patients.Their nuclear acid changes were:c.C473T and c.C397T,leading to amino acid change accordingly:p.T158M and p.R133C.There were no same mutations from their parents.Phenotype of patient with c.C397T was milder than patient with c.C473T.Conclusions Most of typical Rett syndrome patients had mutations in MECP2 gene.Gene test should be performed.Their biological parents should be detected accordingly if the patient had positive found to support genetic counseling.

The effect of NaAsO2 on the binding of methyl CpG binding protein 2, DNA methyltransferase 1 and histone deacetylase 1 to the promoter of MGMT gene in HaCaT cells / 中国地方病学杂志

Objective To investigate the effect of NaAsO2 on the binding levels of methyl CpG binding protein 2(MeCP2),DNA methyltransferase 1 (DNMT1) and histone deacetylase 1(HDAC1) to the hypermethylation promoter region of MGMT gene in HaCaT cells,in order to provide a basis to deepen the interpretation of the role of arsenic poisoning mechanism.Methods HaCaT cells were treated repeatedly and interval with different concentrations of NaAsO2(3.13,6.25,12.50,25.00 μnol/L,respectively) for 72 h.Untreated HaCaT was used as blank control group and human epidermal squamous carcinoma cell line(A431 cells) as positive control group.The binding levels to the two transcription regulatory regions(ChIP1,ChIP2) and to the coding region(ChIP3) of MGMT 8ene were detected by chromatin immuno-precipitation combined with quantitative PCR.Results The differences of binding levels of MeCP2,DNMT1 and HDAC1 to ChIP1 and ChIP2 in each group were significant (F=7.387,84.634,78.442 and 19.263,69.649,26.546,all P ＜ 0.05).The binding levels of MeCP2,DNMT1 and HDAC1 to ChIP1 and ChIP2 in each NaAsO2 exposed group[3.13 μmol/L NaAsO2 exposed group:(136.00 ±16.97)％,(145.00 ± 2.83)％,(88.50 ± 19.09)％ and (106.50 ± 37.48)％,(112.34 ± 8.73)％,(59.71 ± 8.49)％;6.25 μmol/L NaAsO2 exposed group:(130.00 ± 42.43)％,(154.50 ± 4.95)％,(101.00 ± 1.27)％ and (88.50 ±3.54)％,(134.32 ± 2.82)％,(102.75 ± 19.91)％ ; 12.50 μmol/L NaAsO2 exposed group:(141.50 ± 23.33)％,(161.50 ± 7.78)％,(125.00 ± 11.31)％ and (119.50 ± 24.75)％,(171.59 ± 3.54)％,(167.61 ± 10.61)％; 25.00μmol/L NaAsO2 exposed group:(134.50 ± 43.13)％,(472.50+ 50.20)％,(383.50 ± 30.41)％ and (180.09 ±12.73)％,(348.50 ± 27.58)％,(158.45 ± 12.02)％] were higher than that in the blank control group[(51.50 ±9.19)％,(82.00 ± 12.73)％,(25.03 ± 2.91)％ and (37.02 ± 4.24)％,(91.56 ± 26.16)％,(19.09 ± 2.90)％,all P ＜ 0.05].The differences of binding levels of MeCP2 to ChIP3 in each group were not significant(F =1.670,P ＞0.05),but the differences of binding levels of DNMT1 and HDAC1 to ChIP3 were significant (F =4.404,9.863,all P ＜ 0.05),and only the binding levels in the 25.00 μmol/L NaAsO2 exposed group [(615.85 ± 29.63)％,(306.09 ± 59.40)％] were higher than that in the blank control group[(99.70 ± 12.02)％,(92.45 ± 48.79)％,all P ＜ 0.05].Conclusions MeCP2 can bind to the methylated MGMT gene transcriptional regulatory regions which are induced by arsenic and leads to histone deacetylation by the recruitment of DNMT1 and HDAC1 and,meanwhile,DNMT1 can bind to the coding region of MGMT gene to recruit HDAC1 in a methyl DNA binding protein(MBD) independence manner and media MGMT gene silencing through the chromatin remodeling way,which might be the early molecular events of arsenic poisoning.

Folate inhibits methyl-CpG-binding protein 2 expression and cervical cancer cell proliferation / 肿瘤研究与临床

Objective To explore the impact of folate on MeCP2 expression and cervical cancer cells growth.Methods Cervical cancer cell lines Caski (HPV16-positive) and C33A (HPV-negative) were treated with different concentrations of folate.MTT,flow cytometry,Western blott and real-time PCR were used to detect the cells’ viability,apoptosis,the expression of MeCP2 protein and mRNA expressions respectively.Results The inhibitions of both cell growth were upgraded with the folate concentration increasing.The differences were significant between the experimental groups and the control group.With increasing of folate concentration,apoptosis ratio of C33A and Caski increased gradually (C33A:r =0.965,P ＜ 0.001; Caski:r =0.973,P ＜ 0.001) and the expression of MeCP2 protein downgraded gradually,presenting significantly negative correlations between them (C33A:r =-0.952,P ＜ 0.001; Caski:r =-0.947,P ＜ 0.001).There was significantly difference for mRNA expression in different concentration groups of Caski and C33A (C33A:F =77.041,P ＜ 0.001; Caski:F =59.885,P ＜ 0.001).In the same concentration group,the expression of MeCP2 protein and mRNA were higher in Caski than that of C33A,and the difference was significant in the concentration of 500 μg/ml group.There was a negative correlation between the expression of MeCP2 protein and cells’ apoptosis ratio (C33A:r =-0.970,P ＜ 0.001; Caski:r =-0.93,P ＜ 0.001).Conclusion Folic acid can inhibit the growth of cerical cancer cells,promote apoptosis and reduce the expression of MeCP2.The aberrant high-expression of MeCP2 can inhibit apoptosis of Caski and C33A.

Research progress of Rett syndrome causing gene MECP2-The structure, function and modulation of MECP2 / 北京大学学报(医学版)

SUMMARY Rett syndrome (RTT) , an X-linked dominant neurodevelopmental disorder characterized by regression of language, stereotype hand movement and loss of purposeful hand use, is primarily caused by mutation of menthyl-CpG-binding protein 2 ( MECP2 ). The 76 kb human MECP2 is characterized by three sab'ent features; a very large intron 2 (60 kb) , an 8. 5 kb 3'-UTR with highly conserved regions and different polyadenylation sites, and a 40 kb intergenic region separating MECP2 from the nearest upstream gene. There are two isoforms of MeCP2, MeCP2el and MeCP2e2. The differences between the two isoforms, the function of the 3'-UTR and the long-range cis-regulatory sequences in the intergenic region were extensively studied. In contrast to initial report, recent studies show that MeCP2 binds not only to methylated promoters and silence transcription, but also to the sites outside of genes containing only a few of CpG islands. Furthermore, MeCP2 can function as both an activator and a repressor of transcription.

Research progress of Rett syndrome causing gene MECP2——The structure,function and modulation of MECP2 / 北京大学学报(医学版)

Rett syndrome(RTT),an X-linked dominant neurodevelopmental disorder characterized by regression of language,stereotype hand movement and loss of purposeful hand use,is primarily caused by mutation of menthyl-CpG-binding protein 2(MECP2).The 76 kb human MECP2 is characterized by three salient features: a very large intron 2(60 kb),an 8.5 kb 3′-UTR with highly conserved regions and different polyadenylation sites,and a 40 kb intergenic region separating MECP2 from the nearest upstream gene.There are two isoforms of MeCP2,MeCP2e1 and MeCP2e2.The differences between the two isoforms,the function of the 3′-UTR and the long-range cis-regulatory sequences in the intergenic region were extensively studied.In contrast to initial report,recent studies show that MeCP2 binds not only to methylated promoters and silence transcription,but also to the sites outside of genes containing only a few of CpG islands.Furthermore,MeCP2 can function as both an activator and a repressor of transcription.Abstract:SUMM ARY Rett syndrome(RTT),an X-linked dom inant neurodevelopmental d isorder characterized by regression of language,stereotype hand movement and loss of purposeful hand use,is primarily caused by mutation of menthyl-CpG-bind ing protein 2(MECP2).The 76 kb humanMECP2is characterized by three salient features: a very large intron 2(60 kb),an 8.5 kb 3′-UTR with highly conserved regions and d ifferent polyadenylation sites,and a 40 kb intergenic region separatingMECP2from the nearest up-stream gene.There are two isoforms ofMeCP2,MeCP2e1 and MeCP2e2.The d ifferences between the two isoforms,the function of the 3′-UTR and the long-range cis-regulatory sequences in the intergenic re-gion were extensively stud ied.In contrast to initial report,recent stud ies show thatMeCP2 binds not only to methylated promoters and silence transcription,but also to the sites outside of genes containing only a few of CpG islands.Furthermore,MeCP2 can function as both an activator and a repressor of transcrip-tion.

A Case of Rett Syndrome with MECP2 Gene Mutation

Rett syndrome is an X-linked dominant, progressive neurodevelopmental disorder, with a prevalence estimated to be one in 10,000-15,000 girls, which is thought to be the second most common genetic causes of mental retardation in females after Down syndrome. Patients with classic Rett syndrome show an apparently normal neonatal period, followed by developmental regression and deceleration of head growth, accompanied by gradual loss of speech and purposeful hand use, and development of microcephaly, seizures, autism, ataxia, intermittent hyperventilation and stereotypic hand movements. After regression between infancy and the fifth year of life, the clinical course stabilizes and patients usually survive into adulthood. It was recently discovered that Rett syndrome is caused by mutations in the methyl-CpG binding protein 2(MECP2) gene. Diagnosis of Rett syndrome is clinically difficult before three years of age, especially in atypical cases, but molecular analysis of the MECP2 gene could assist correct diagnosis in some patients. Recently, we diagnosed a case of Rett syndrome in a two year-old girl by mutational analysis of the MECP2 gene and want to report this case with brief review of literature.