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MicroRNA-23a/b and microRNA-27a/b suppress Apaf-1 protein and alleviate hypoxia-induced neuronal apoptosis.

Chen Q, Xu J, Li L, Li H, Mao S, Zhang F, Zen K, Zhang CY, Zhang Q - Cell Death Dis (2014)

Bottom Line: In the present study, we found that four microRNAs (miR-23a/b and miR-27a/b) of miR-23a-27a-24 and miR-23b-27b-24 clusters play key roles in modulating the expression of Apaf-1.First, we found that miR-23a/b and miR-27a/b suppressed the expression of Apaf-1 in vitro.In conclusion, our results demonstrate, in central neural system, that miR-23a/b and miR-27a/b are endogenous inhibitory factors of Apaf-1 expression and regulate the sensitivity of neurons to apoptosis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu, China.

ABSTRACT
Expression of apoptotic protease activating factor-1 (Apaf-1) gradually decreases during brain development, and this decrease is likely responsible for the decreased sensitivity of brain tissue to apoptosis. However, the mechanism by which Apaf-1 expression is decreased remains elusive. In the present study, we found that four microRNAs (miR-23a/b and miR-27a/b) of miR-23a-27a-24 and miR-23b-27b-24 clusters play key roles in modulating the expression of Apaf-1. First, we found that miR-23a/b and miR-27a/b suppressed the expression of Apaf-1 in vitro. Interestingly, the expression of the miR-23-27-24 clusters in the mouse cortex gradually increased in a manner that was inversely correlated with the pattern of Apaf-1 expression. Second, hypoxic injuries during fetal distress caused reduced expression of the miR-23b and miR-27b that was inversely correlated with an elevation of Apaf-1 expression during neuronal apoptosis. Third, we made neuronal-specific transgenic mice and found that overexpressing the miR-23b and miR-27b in mouse neurons inhibited the neuronal apoptosis induced by intrauterine hypoxia. In conclusion, our results demonstrate, in central neural system, that miR-23a/b and miR-27a/b are endogenous inhibitory factors of Apaf-1 expression and regulate the sensitivity of neurons to apoptosis. Our findings may also have implications for the potential target role of microRNAs in the treatment of neuronal apoptosis-related diseases.

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The expression of miR-23-27 clusters increases during brain development. (a) Schematic of the Apaf-1 3′UTR indicating the locations of the miR-23 and miR-27 target sites that are conserved in vertebrates. The Apaf-1 3′UTR contains evolutionarily well-conserved sequences matched for the miR-23 and miR-27 families that were predicted by computer-aided algorithms. The free energies (mfes) of microRNA bindings were calculated by RNAHybrid software (BiBiServ, Bielefeld, Germany). (b) Quantitative RT-PCR detection of miR-23a, miR-23b, miR-27a, miR-27b, and miR-24 in cerebral cortex samples at different developmental stages (n=5, one-way ANOVA with Newman–Keuls multiple comparison test, *P<0.05, **P<0.01, and ***P<0.001). (c) Dot blot analyses of miR-23b and U6 snRNA in total RNA of the cerebral cortex. Each lane was loaded with equal amounts of total RNA extracted from the mixed samples of five individuals. (d) Expression of miR-23b in cortices of E18 and adult brain tissues detected by in situ hybridization. Scale bar represents 250 μm; scale bar (insert) represents 200 μm
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fig2: The expression of miR-23-27 clusters increases during brain development. (a) Schematic of the Apaf-1 3′UTR indicating the locations of the miR-23 and miR-27 target sites that are conserved in vertebrates. The Apaf-1 3′UTR contains evolutionarily well-conserved sequences matched for the miR-23 and miR-27 families that were predicted by computer-aided algorithms. The free energies (mfes) of microRNA bindings were calculated by RNAHybrid software (BiBiServ, Bielefeld, Germany). (b) Quantitative RT-PCR detection of miR-23a, miR-23b, miR-27a, miR-27b, and miR-24 in cerebral cortex samples at different developmental stages (n=5, one-way ANOVA with Newman–Keuls multiple comparison test, *P<0.05, **P<0.01, and ***P<0.001). (c) Dot blot analyses of miR-23b and U6 snRNA in total RNA of the cerebral cortex. Each lane was loaded with equal amounts of total RNA extracted from the mixed samples of five individuals. (d) Expression of miR-23b in cortices of E18 and adult brain tissues detected by in situ hybridization. Scale bar represents 250 μm; scale bar (insert) represents 200 μm

Mentions: Compared with the downregulation of the mRNA level, the decrease of Apaf-1 protein level was more dramatic, and this indicates that Apaf-1 gene expression might be regulated at the posttranscriptional level. The miRNAs are a type of endogenous small RNA that suppresses gene expression at the posttranscriptional level. Using computer-aided algorithms,26 we found that four (miR-23a, miR-23b, miR-27a, and miR-27b) of the miR-23-27-24 clusters had conserved putative binding sites for Apaf-1 mRNA on 3′-UTR (Figure 2a). The miR-24 had less conserved binding sites. In the mouse genome, the mmu-miR-23a-27a-24-2 cluster is located on chromosome 8, and the mmu-miR-23b-27b-24-1 cluster is located on chromosome 13. We examined the levels of miR-23a/b, miR-27a/b, and miR-24 expression at the same developmental time points. The results from quantitative reverse transcription-PCR (qRT-PCR) experiments showed that expression of the miR-23-27-24 clusters clearly increased over these time points. The miR-23b expression gradually increased during maturation of the brain, and the miR-23b transcript levels were approximately fivefold greater in adult mouse cortices than in the cortices of the E18 pups. The miR-27b also exhibited dramatically increased expression in the adult stage. The miR-23a and miR-27a also showed gradually increases in expression, but these increases were relatively mild compared with that of the miR-23b-27b cluster. The miR-24 had a significantly increased expression pattern during development (Figure 2b). To further corroborate the qRT-PCR results, a RNA dot blotting assay and in situ hybridization staining were employed. As shown in Figure 2c, miR-23b levels were much higher in P60 adult mouse cortices than in E18 pup cortices. In contrast to IHC staining signal of Apaf-1, the in situ hybridization results revealed that miR-23b was abundantly expressed in the neurons and more miR-23b signals were detected in adult brain neurons (Figure 2d). Therefore, the expression patterns of the Apaf-1 gene and the miR-23-27 clusters were inversely correlated that indicates that the miR-23-27 clusters might inhibit Apaf-1 gene expression during brain development.


MicroRNA-23a/b and microRNA-27a/b suppress Apaf-1 protein and alleviate hypoxia-induced neuronal apoptosis.

Chen Q, Xu J, Li L, Li H, Mao S, Zhang F, Zen K, Zhang CY, Zhang Q - Cell Death Dis (2014)

The expression of miR-23-27 clusters increases during brain development. (a) Schematic of the Apaf-1 3′UTR indicating the locations of the miR-23 and miR-27 target sites that are conserved in vertebrates. The Apaf-1 3′UTR contains evolutionarily well-conserved sequences matched for the miR-23 and miR-27 families that were predicted by computer-aided algorithms. The free energies (mfes) of microRNA bindings were calculated by RNAHybrid software (BiBiServ, Bielefeld, Germany). (b) Quantitative RT-PCR detection of miR-23a, miR-23b, miR-27a, miR-27b, and miR-24 in cerebral cortex samples at different developmental stages (n=5, one-way ANOVA with Newman–Keuls multiple comparison test, *P<0.05, **P<0.01, and ***P<0.001). (c) Dot blot analyses of miR-23b and U6 snRNA in total RNA of the cerebral cortex. Each lane was loaded with equal amounts of total RNA extracted from the mixed samples of five individuals. (d) Expression of miR-23b in cortices of E18 and adult brain tissues detected by in situ hybridization. Scale bar represents 250 μm; scale bar (insert) represents 200 μm
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Related In: Results  -  Collection

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fig2: The expression of miR-23-27 clusters increases during brain development. (a) Schematic of the Apaf-1 3′UTR indicating the locations of the miR-23 and miR-27 target sites that are conserved in vertebrates. The Apaf-1 3′UTR contains evolutionarily well-conserved sequences matched for the miR-23 and miR-27 families that were predicted by computer-aided algorithms. The free energies (mfes) of microRNA bindings were calculated by RNAHybrid software (BiBiServ, Bielefeld, Germany). (b) Quantitative RT-PCR detection of miR-23a, miR-23b, miR-27a, miR-27b, and miR-24 in cerebral cortex samples at different developmental stages (n=5, one-way ANOVA with Newman–Keuls multiple comparison test, *P<0.05, **P<0.01, and ***P<0.001). (c) Dot blot analyses of miR-23b and U6 snRNA in total RNA of the cerebral cortex. Each lane was loaded with equal amounts of total RNA extracted from the mixed samples of five individuals. (d) Expression of miR-23b in cortices of E18 and adult brain tissues detected by in situ hybridization. Scale bar represents 250 μm; scale bar (insert) represents 200 μm
Mentions: Compared with the downregulation of the mRNA level, the decrease of Apaf-1 protein level was more dramatic, and this indicates that Apaf-1 gene expression might be regulated at the posttranscriptional level. The miRNAs are a type of endogenous small RNA that suppresses gene expression at the posttranscriptional level. Using computer-aided algorithms,26 we found that four (miR-23a, miR-23b, miR-27a, and miR-27b) of the miR-23-27-24 clusters had conserved putative binding sites for Apaf-1 mRNA on 3′-UTR (Figure 2a). The miR-24 had less conserved binding sites. In the mouse genome, the mmu-miR-23a-27a-24-2 cluster is located on chromosome 8, and the mmu-miR-23b-27b-24-1 cluster is located on chromosome 13. We examined the levels of miR-23a/b, miR-27a/b, and miR-24 expression at the same developmental time points. The results from quantitative reverse transcription-PCR (qRT-PCR) experiments showed that expression of the miR-23-27-24 clusters clearly increased over these time points. The miR-23b expression gradually increased during maturation of the brain, and the miR-23b transcript levels were approximately fivefold greater in adult mouse cortices than in the cortices of the E18 pups. The miR-27b also exhibited dramatically increased expression in the adult stage. The miR-23a and miR-27a also showed gradually increases in expression, but these increases were relatively mild compared with that of the miR-23b-27b cluster. The miR-24 had a significantly increased expression pattern during development (Figure 2b). To further corroborate the qRT-PCR results, a RNA dot blotting assay and in situ hybridization staining were employed. As shown in Figure 2c, miR-23b levels were much higher in P60 adult mouse cortices than in E18 pup cortices. In contrast to IHC staining signal of Apaf-1, the in situ hybridization results revealed that miR-23b was abundantly expressed in the neurons and more miR-23b signals were detected in adult brain neurons (Figure 2d). Therefore, the expression patterns of the Apaf-1 gene and the miR-23-27 clusters were inversely correlated that indicates that the miR-23-27 clusters might inhibit Apaf-1 gene expression during brain development.

Bottom Line: In the present study, we found that four microRNAs (miR-23a/b and miR-27a/b) of miR-23a-27a-24 and miR-23b-27b-24 clusters play key roles in modulating the expression of Apaf-1.First, we found that miR-23a/b and miR-27a/b suppressed the expression of Apaf-1 in vitro.In conclusion, our results demonstrate, in central neural system, that miR-23a/b and miR-27a/b are endogenous inhibitory factors of Apaf-1 expression and regulate the sensitivity of neurons to apoptosis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu, China.

ABSTRACT
Expression of apoptotic protease activating factor-1 (Apaf-1) gradually decreases during brain development, and this decrease is likely responsible for the decreased sensitivity of brain tissue to apoptosis. However, the mechanism by which Apaf-1 expression is decreased remains elusive. In the present study, we found that four microRNAs (miR-23a/b and miR-27a/b) of miR-23a-27a-24 and miR-23b-27b-24 clusters play key roles in modulating the expression of Apaf-1. First, we found that miR-23a/b and miR-27a/b suppressed the expression of Apaf-1 in vitro. Interestingly, the expression of the miR-23-27-24 clusters in the mouse cortex gradually increased in a manner that was inversely correlated with the pattern of Apaf-1 expression. Second, hypoxic injuries during fetal distress caused reduced expression of the miR-23b and miR-27b that was inversely correlated with an elevation of Apaf-1 expression during neuronal apoptosis. Third, we made neuronal-specific transgenic mice and found that overexpressing the miR-23b and miR-27b in mouse neurons inhibited the neuronal apoptosis induced by intrauterine hypoxia. In conclusion, our results demonstrate, in central neural system, that miR-23a/b and miR-27a/b are endogenous inhibitory factors of Apaf-1 expression and regulate the sensitivity of neurons to apoptosis. Our findings may also have implications for the potential target role of microRNAs in the treatment of neuronal apoptosis-related diseases.

Show MeSH
Related in: MedlinePlus