Abstract
Cerebral palsy is a group of non-progressive motor impairment syndromes caused by brain lesions during development. Herein, we investigated the relationship between nucleotide variations in a miRNA coding region and the predisposition of Chinese children to cerebral palsy. A total of 233 CP patients and 256 healthy participants were enrolled, and 60 children were selected from each group for plasma miRNA detection. We screened the coding regions of pri-miR-124-1, -2, and -3 using PCR and sequencing. The expression of miR-124 was determined by qRT-PCR. Luciferase assays and Western blots were used to confirm the regulation of target genes by miR-124. The function of miR-124 was further identified in SH-SY5Y cells by detecting cell viability and apoptosis. We revealed that the rare alleles T of rs3802169 and G of rs191727850 were found to be associated with an increased risk of cerebral palsy (OR=3.71, 95% CI 1.74–7.92 and OR=2.18, 95% CI 1.36–3.49, respectively). We further showed that the levels of mature miR-124 were down-regulated by the C-to-T variation in vitro. More importantly, the reduction of miR-124 resulting from the C-to-T change led to the less-efficient inhibition of the target genes ITGB1, LAMC1 and BECN1, which may play important roles during the development of the nervous system. Meanwhile, the reduction in the expression of miR-124 was also related to the increased nuclear translocation of apoptosis-inducing factor (AIF) under oxidative stress, thereby inducing more cell apoptosis. Our results suggest that one functional polymorphism in pri-miR-124-1 might contribute to the genetic predisposition of Chinese children to cerebral palsy by disrupting the production of miR-124, which consequently interfered in the expression and function of the target genes of miR-124.
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Introduction
Cerebral palsy (CP) is a group of non-progressive motor impairment syndromes caused by lesions of the brain that arise early in development1. Risk factors that lead to this condition such as preterm birth, intrauterine infection, intrauterine growth restriction, and perinatal stroke have been identified2,3. Furthermore, genetic makeup is also related to the predisposition to cerebral damage by influencing the response to the presence of these risk factors4,5. Several genetic variants that cause Mendelian forms of CP have been described by other researchers6, and variants in the IL-6 coding region have also been reported to contribute to enhanced genetic predisposition to CP7.
MicroRNAs are a group of negative regulators of gene expression that repress gene expression by targeting the 3′UTR of mRNA. A group of miRNAs expressed in the central nervous system are dynamically regulated both during physiological brain development and in vitro neural differentiation of stem cells, indicating that they have a significant contribution to neural development and function8,9. Meanwhile, abnormal miRNA levels have been found to be related to human nervous system diseases such as NTD, schizophrenia, and Alzheimer's disease10,11,12. miR-124 is a brain-enriched miRNA that is derived from three independent genes (miR-124-1, -2, and -3) located on chromosome 8 or 20. Interestingly, knock-out of just the miR-124-1 gene in mice resulted in a significant reduction of the mature miR-124 level, defective neuronal survival, and axonal outgrowth, as well as smaller brain size13. Jenn-Yah et al have reported that the miR-124 gene is one of the most abundantly expressed in the adult nervous system and that it regulates neurite outgrowth during neuronal differentiation14. However, it remains unknown whether the SNPs of miR-124 affect miRNA biogenesis and human genetic disease. BECN1 mRNA and protein are abundantly expressed in neurons and glia in human brains15,16 . Beclin-1 is a protein encoded by the BECN1 gene. It regulates the autophagy-induced activity of Vps3417 and plays an important role in the formation of autophagosomes18. The integrin β1 gene (ITGB1) encodes β1 integrin, which could also promote neurite outgrowth19. The laminin c1 gene (LAMC1) could encode ECM-associated proteins. In addition, mutations in ECM-associated proteins lead to cortical neuron lamination defects, including fragility of the basement membrane and detachment of radial glia from the basement membrane20. In this study, BECN1, ITGB1 and LAMC1 genes were selected as the target genes to predict the functional consequence of enhanced processing of miR-124.
Increasing evidence has shown that single nucleotide polymorphisms (SNPs) or mutations in miRNA coding regions may alter miRNA expression and/or maturation and be related to the occurrence of diseases21,22. Meanwhile, some of the alterations in expression are detectable in serum23. In this study, to explore the relationship between SNPs in the miRNA coding region and the occurrence of CP, we detected the serum levels of 11 candidate miRNAs in 56 CP patients. The coding regions of 3 altered miRNAs were sequenced, and one SNP was found to be related to the genetic predisposition to CP by reducing the level of mature miRNA.
Materials and methods
Subjects
In this study, 233 CP patients (103 girls and 130 boys with mean ages±SD of 18.9±13.8 months) were chosen from the Rehabilitation Center of the Affiliated Yantai Yuhuangding Hospital of Qingdao University from 30th March 2012 to 28th September 2015. In addition, 256 healthy control participants (112 girls and 144 boys with mean ages±SD of 19.2±11.7 months) were chosen from Department of Pediatrics at the same hospital. Of these, 60 CP patients and 60 control patients were selected for plasma miRNA detection. The age of the selected children ranged from 5 months to 36 months. All subjects were Han Chinese from North China, and written informed consent to participate in this study was provided by the children's parents. Child neurologists diagnosed and classified the CP by clinical examination or by using medical records, including brain imaging, according to the guidelines proposed by the Surveillance of CP in Europe network24. Children suffering from hypotonia, ataxia, myopathy, a genetic syndrome or a chromosomal anomaly were excluded. Blood samples were collected in tubes containing EDTA by skilled nurses on the second day of hospitalization. Plasma was separated from the 500 μL blood sample by centrifugation (1500 ×g for 15 min) at 4 °C.
DNA extraction and genotyping
Genomic DNA was extracted from peripheral blood samples from the CP patients and the controls by using the QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. DNA quantity and quality were determined using NanoDrop 2000 ultraviolet-visible spectrophotometry (Thermo Fisher Scientific, Wilmington, DE, USA). The PCR products were sequenced in the forward direction with the ABI 3730xl sequencing platform. The sequencing results were analyzed by using DNAMAN and BLAST. The primers were as follows: miR-124-1 forward 5′--3′, reverse 5′--3′ and miR-124-2 forward 5′--3′, reverse 5′--3′. The genomic DNA fragment amplified by the primers contains a single nucleotide polymorphism (SNP). The SNP genotypes of the genomic DNA samples were determined by sequence analysis.
RNA isolation
Total plasma RNA was extracted using the TRIzol LS reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. Briefly, 250 μL of plasma was added to 750 μL of TRIzol reagent and incubated at room temperature for 5 min. Chloroform was added to the samples, and they were vigorously mixed and incubated at room temperature for 5 min. Following incubation, the samples were centrifuged at 12 000 ×g for 15 min at 4 °C. RNA was precipitated from the aqueous phase by addition of isopropyl alcohol to a fresh tube containing the supernatant in the aqueous phase. The integrity of the RNA was tested by spectroscopic analysis.
RNA secondary structure prediction
The secondary structure of the 316-bp pri-miR-124 including the mutation site was predicted using the RNAfold web server (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi).
miR-124 expression vectors
To construct the miR-124 expression vectors, we engineered expression plasmids with 425 nt of pre-miR-124-1 and its flanking regions, which included two SNP sites. The pri-miR-124-1 DNA sequences were amplified through PCR from the cDNA of different genotypes. The following primers of pri-miR-124-1 were used: forward 5′-′ and reverse 5′--3′. The following primers of pri-miR-124-2 were used: forward 5′--3′ and reverse 5′--3′. The PCR products were cloned into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA). The sequences of these vectors were confirmed by direct sequencing with the ABI 3730xl sequencing platform.
Real-time reverse transcriptase quantitative PCR
The expression levels of miR-124-3p and -5p and the plasma levels of the miR-124 genes were determined by qRT-PCR using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) together with miRNA-specific TaqMan MGB probes. U6 snRNA was used for normalization. Each sample in each group was measured in triplicate, and the experiment was repeated at least three times.
Dual luciferase assay
The full length of the 1031-bp BECN1 3′UTR, the full length of the 2879-bp LAMC1 3′UTR and a 668-bp segment of ITGB1 were cloned separately into the pmirGLO vector downstream of the firefly luciferase coding region (Promega, Madison, WI, USA) to generate luciferase reporter vectors. For the luciferase reporter assays, HEK293T cells were seeded in 48-well plates. miR-124 expression vectors and luciferase reporter vectors were co-transfected by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and different genotypes of pri-miR-124 (CC, CT or TT). The cells were harvested 48 h after transfection, and the luciferase activity was assayed using the Dual-Luciferase Assay kit (Promega, Madison, WI, USA). Each treatment was performed in triplicate in three independent experiments. The results were expressed as relative luciferase activity (Firefly LUC/Renilla LUC).
Western blot
The proteins were extracted with an appropriate amount of protein lysis buffer (Beyotime, Shanghai, China) in each plate. After centrifugation at 15 000 ×g and 4 °C, the supernatant was collected. A 25% volume of 5× loading buffer was added to the protein samples before the protein was loaded, and the samples were denatured at 100 °C for 5 min. Equal amounts of protein (20 μg) were dissolved using 12% SDS-PAGE and then transferred onto PVDF membranes, followed by the addition of blocking buffer (TBST containing 5% bovine serum albumin) at room temperature for 1 h. After that, the PVDF membranes were incubated with the appropriate primary antibodies overnight at 4 °C, and the membranes were washed 3 times with Tris-buffered saline with Tween-20 (TBST) followed by an incubation for 1 h with HRP-conjugated secondary antibody at room temperature. The PVDF membranes were developed by an enhanced chemiluminescent (ECL) system (Li-cor, Lincoln, NE, USA) and exposed to UVP chemiluminescence (Biolite LLC, Upland, CA, USA). Protein quantification was performed by AlphaView SA software by calculating the band densities.
Apoptosis analysis
Cell apoptosis was detected using annexin V-FITC and propidium iodide (PI) staining and analyzed by flow cytometry. Briefly, the SH-SY5Y cells (∼1×105) were transfected with pri-miR-124-CC, -CT or -TT expression vectors for 48 h, while SH-SY5Y cells in control group was only treated with DMEM medium. Then, the cells were harvested, washed with PBS solution, cultured with annexin V-FITC and PI at room temperature for 15 min in the dark, and analyzed using a FACS Calibur (BD Bioscience) with a minimum of 10 000 cells in each sample.
Cell proliferation assay
Cell viability was determined through MTT assay. Briefly, human neuroblastoma SH-SY5Y cells were seeded in 96-well plates at a low density of 5×103 cells per well in DMEM culture and allowed to attach overnight. The cells were then transfected with different genotype mir-124 expression vectors for 48 h at 37 °C in 5% CO2. Then, 20 μL of MTT (5 mg/mL) (Sigma, St Louis, MO, USA) was added into each well at 4 different time points (0 h, 24 h, 48 h, 72 h) after transfection, and the cells were incubated for another 4 h. The optical density was recorded at 570 nm after the DMSO addition using a microplate reader (Spectra Max M2, Molecular Devices, USA).
Oxidative stress assay
To mimic oxidative stress conditions, SH-SY5Y cells were transfected with miR-124 inhibitor for 24 h and then treated with 0.5 mmol/L H2O2 for 1 h at 37 °C. The cells were centrifuged and washed to remove the hydrogen peroxide. Then, 10 μL of annexin V-FITC and 5 μL of PI were added, and the cells were cultured in the dark at 4 °C before being analyzed using a FACS Calibur (BD Bioscience, USA).
ELISA
The plasma expression levels of the BECN1 protein were determined by enzyme-linked immunosorbent assay (ELISA) kits (eBioscience Inc, San Diego, CA, USA) following the manufacturers' instructions.
Statistical analysis
For gene analysis, a test for Hardy-Weinberg equilibrium was performed on the allele and genotype frequency analysis using the SHEsis online software platform (http://analysis.bio-x.cn). The relative risk was approximated by the estimate of the odds ratio (OR). All ORs were adjusted for age and sex using logistic regression models. The tested correlation between BECN1 levels and miR-124 expression was analyzed using χ2-analysis. Independent analysis of the differences between groups was performed with t-tests. The data are reported as the mean±SD. Comparisons between multiple groups were performed by one-way ANOVA followed by the Student–Newman–Keuls (SNK) method. All statistical analyses were analyzed with the SPSS Statistical Package version 16. P<0.05 was considered statistically significant.
Results
The genotypic distribution of SNPs in CP patients and controls
miR-124 is a brain-enriched miRNA, the aberrant expression of which contributes to pathological conditions involving the central nervous system. To determine whether there are relationships between CP genetic predisposition and nucleotide variations in the miR-124 coding region, we first screened the sequences of pri-miR-124-1, -2 and -3 in 233 CP patients and 256 healthy controls. Differences in allele frequencies and genotype frequencies between CP patients and healthy controls were calculated (Table 1). As shown in Table 1, the distribution of allele frequencies and genotype frequencies of SNPs rs531564, rs73662598 and rs569595042 in the controls and patients was in Hardy-Weinberg equilibrium (P>0.05). Significant differences were found in the allele and genotype distribution of rs3802169 and rs191727850 within pri-miR-124-1 and pri-miR-124-2 in CP patients compared with controls (P<0.05). Meanwhile, we concluded that C/T heterozygosity of rs3802169 (OR=3.60, 95%CI=1.65–7.82) and A/G heterozygosity of rs191727850 (OR=1.93, 95%CI=1.16–3.19) were associated with an increased risk of CP.
Prediction of the effects of SNPs rs3802169 and rs191727850 on the structure of pri-miR-124-1 and -2
To understand the functions of these two SNPs, we first analyzed the predicted secondary structure and molecular stability of pri-miR-124-1 and -2. As shown in Figure 1A, the rare allele T of rs3802169 at the position -78 relative to pre-miR-124-1 causes an apparent change in the secondary structure and a lower predicted ΔG from -113.80 kcal/mol to -114.60 kcal/mol. Furthermore, the expression of mature miR-124-3p and -5p was up-regulated in the HEK293T cells transfected with the pri-miR-124-1-TT expression vector (Figure 1B). The rare allele G of rs191727850 at the position of -62 relative to pre-miR-124-2 did not change the predicted secondary structure of pri-miR-124-2 nor the predicted ΔG (Figure 2A). In addition, the relative miR-124-3p and -5p expression levels in the AA, AG and GG genotype groups are shown in Figure 2B. We concluded that there were no significant differences in the expression levels of mature miR-124-3p and -5p after transfection with AA, AG and GG genotype pri-miR-124-1 expression vectors (Figure 2B).
Prediction of miR-124-3p and relative luciferase activity in different groups
Since the biological function of a miRNA mostly depends on what downstream genes it represses, we predicted the direct targets of miR-124-3p using the online bioinformatics tool TargetScan (http://www.targetscan.org). As shown in Figure 3A, miR-124-3p may repress BECN1 expression by targeting a 7-mer site in the 3′UTR of BECN1 mRNA. Subsequently, we constructed a dual-luciferase reporter vector using the full length of the BECN1 3′UTR as the 3′UTR of firefly luciferase. The reporter vector was co-transfected with the miR-124-3p mimic or inhibitor into HEK293T cells. The cells were lysed 48 h after transfection, and the luciferase activity was detected. As shown in Figure 3B, the relative luciferase activity was reduced by 45.2% by the miR-124-3p mimic and increased by 13.4% in the cells transfected with the miR-124-3p inhibitor. These results indicated that miR-124-3p repressed the luciferase activity by targeting the 3′UTR of BECN1. To examine whether endogenous BECN1 expression was repressed by miR-124-3p, HEK293T cells were transfected with miR-124-3p mimic or inhibitor. The cells were lysed 48 h after transfection, and the BECN1 expression level was determined by immunoblotting. As shown in Figure 3C, the BECN1 protein level was markedly reduced in cells transfected with the miR-124-3p mimic and up-regulated in cells transfected with the miR-124-3p inhibitor, which was consistent with the result of the dual-luciferase analysis.
To initiate a study of the functional consequence of enhanced processing of miR-124 for its target gene, we chose three miR-124-3p target genes. Among them, LAMC1 and ITGB1 were identified as endogenous targets of miR-124, both of which are highly expressed by neural progenitors and are related to neuronal differentiation. The full length LAMC1 3′UTR and a 668-bp segment of the ITGB1 3′UTR were separately cloned into the pmirGLO vector downstream of the firefly luciferase coding region to generate luciferase reporter vectors. These constructed reporters were transiently transfected into HEK293T cells together with an expression plasmid containing one genotype of miR-124-1. Compared with cells transfected with pri-miR-124-1-CC, the activity of firefly luciferase was increased by 12.5% (P<0.01) or 47.0% (P<0.01) in cells co-transfected with pmirGLO-LAMC1 and pri-miR-124-1-CT or -TT, respectively. The activity of firefly luciferase was decreased by 40.6% (P<0.01) in cells co-transfected with pmirGLO-ITGB1 and pri-miR-124-1-TT compared to the activity in cells transfected with pri-miR-124-1-TT. Meanwhile, the activity of firefly luciferase was significantly decreased by 20.5% and 39.8% in cells co-transfected with pmirGLO-BECN and pri-miR-124-CT or pri-miR-124-TT, respectively (P<0.05 or P<0.01). These results indicate that the C-to-T change in rs3802169 can inhibit target gene expression by up-regulating miR-124-1.
Expression levels of LC3II and LC3I were determined by Western blot
BECN1 plays an important role during autophagic programmed cell death, and the LC3II/I value was used to evaluate the degree of autophagy. We detected the LC3II and LC3I levels in SH-SY5Y cells transfected with expression vector with different genotypes of pri-miR-124 through Western blot. As shown in Figure 4A, the level of the BECN1 protein continually increased in the cells transfected with pri-miR-124-CC, -CT and -TT. Meanwhile, the level of the LC3I protein was down-regulated in cells transfection with pri-miR-124-CT and -TT compared to the level in those transfected with pri-miR-124-CC. However, the level of the LC3II protein was up-regulated in cells transfected with pri-miR-124, especially pri-miR-124-TT, suggesting an enhanced autophagy pathway.
Cell proliferation and apoptosis analysis of SH-SY5Y cells transfected with miR-124-CC, -CT and -TT
Subsequently, the cell viability and apoptosis of SH-SY5Y cells transfected with expression vectors with different genotypes of pri-miR-124 were examined through MTT assay and flow cytometry. As shown in Figure 4B, the relative viability of SH-SY5Y cells transfected with the pri-miR-124-CC vector was ∼35%, ∼73% and ∼85% at 24 h, 48 h and 72 h, which are higher than the viability of cells treated with the pri-miR-124-CT vector (∼33%, ∼58% and ∼64%, P<0.05). In addition, we found a lower cell viability at 72 h (∼52%, P<0.05) in cells treated with the pri-miR-124-TT vector compared to cells transfected with the pri-miR-124-CC or -CT expression vectors. Flow cytometry revealed that SH-SY5Y cells transfected with pri-miR-124-CT and -TT expression vectors had higher apoptosis rates (∼14.1% and ∼17.3%, P<0.05 or P<0.01, respectively) than cells transfected with the pri-miR-124-CC expression vector (∼9.8%) (Figure 4C).
Plasma miR-124 and BECN1 levels detected by qRT-PCR
To further explore the function of the C-to-T variation in rs3802169 in vivo, the plasma miR-124 and BECN1 levels of CP patients and controls were detected through qRT-PCR and ELISA, respectively. As shown in Figure 5A, the plasma miR-124 level of CP patients was higher than that of the control group (P<0.05). However, the plasma level of the BECN1 protein was lower than that of the control group (P<0.05, Figure 5B). In addition, the result of the correlation analysis indicated a significant negative correlation between serum miR-124 and BECN1 levels in CP patients (Figure 5C).
Expression of AIF and the cell apoptosis analysis in control and with miR-124 inhibitor
Increased oxidants and decreased antioxidants are usually found in children with CP and contribute to the pathogenesis of cerebral injury25. Under oxidative stress, apoptosis-inducing factor (AIF) will translocate from the mitochondria to the nucleus and induce cell apoptosis. To mimic the oxidative conditions, SH-SY5Y cells were transfected with miR-124 inhibitor for 24 h and then treated with 0.5 mmol/L H2O2 for 1 h. As shown in Figure 6A, the AIF level was significant higher in the nucleus when the cells were treated with the miR-124 inhibitor. Meanwhile, the AIF level was reduced in the cytoplasm, and the total AIF expression was not changed. These results indicated that miR-124 inhibitor can up-regulate AIF cytoplasm-nucleus translocation under oxidant stress. The cell apoptosis assay indicated that the apoptotic cell number was markedly up-regulated when the cells were treated with the miR-124 inhibitor compared with the apoptotic cell number of the control (Figure 6B).
Discussion
Cerebral palsy is a group of non-progressive motor impairment syndromes caused by brain lesions during development. Cerebral palsy is caused by abnormal development or damage to the parts of the brain that control movement, balance, and posture. The risk factors for CP are mainly related to the external element, such as intrauterine infection, intrauterine growth restriction and perinatal stroke. However, genetic factors have also recently been confirmed as contributing to the susceptibility to cerebral damage and being related to the pathogenesis of CP. Recently, a genetic association study confirmed that nucleotide variations in protein coding genes are related to CP, such as in IL-6 and CCL18 TNF-α26,27,28. In this study, we screened a more than 400-bp segment of the pri-miR-124-1, -2, -3 coding regions, and established this kind of association by confirming significant differences in the genotype distribution of rs3802169 and rs191727850 in 233 CP patients and 256 healthy controls. The rare alleles T of rs3802169 and G of rs191727850 were found to be associated with an increased risk of CP (OR=3.71, 95%CI 1.74–7.92; OR=2.18, 95%CI 1.36–3.49). Further analysis showed that the levels of mature miR-124 were down-regulated by the C-to-T change in vitro, which was consistent with the in vivo detection that the level of mature miR-124 was lower in 60 CP patients with the CT genotype. We predicted the effect of the SNP rs3802169 on the secondary structure of miR-124. The T allele causes a change in the predicted secondary structure and a lower ΔG compared with those with the C allele. Therefore, we speculated that the SNP rs3802169 might influence the stability of the pri-miR, the efficiency of changing from pri-miR into pre-miR, or the efficiency of processing pre-miR into the mature miR. More importantly, the reduction of miR-124, as a consequence of the C to T change, further led to reduced efficient inhibition of the target genes, ITGB1, LAMC1 and BECN1, which may play important roles during the development of the nervous system. Thus, our data collectively suggested that one functional polymorphism in pri-miR-124-1 might contribute to the genetic predisposition to CP by disrupting the production of miR-124, which consequently interfered in the expression and function of target genes of miR-124.
In this study, we found that enhanced AIF nucleus translocation is related to the miR-124 level reduction under oxidative stress and is related to up-regulated cell apoptosis, which may provide another piece of evidence to explain the relationship between miR-124 and CP.
In conclusion, we established the first association between SNPs in miR-124-1 and the predisposition to CP in one Chinese Han population by identifying one functional SNP in pri-miR-124-1. These findings may give insight into the understanding of RPL development and may create an opportunity to expand the approach to diagnosing and treating RPL.
Abbreviation
AI, apoptosis inducing factor; CP, cerebral palsy; ELISA, enzyme-linked immunosorbent assay; HWE, Hardy-Weinberg equilibrium; OR, odds ratio; SNPs, single nucleotide polymorphisms.
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Li, H., Wang, Xl., Wu, Yq. et al. Correlation of the predisposition of Chinese children to cerebral palsy with nucleotide variation in pri-miR-124 that alters the non-canonical apoptosis pathway. Acta Pharmacol Sin 39, 1453–1462 (2018). https://doi.org/10.1038/aps.2017.211
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DOI: https://doi.org/10.1038/aps.2017.211